Substituted 2-[2-(phenyl) ethylamino] alkaneamide derivatives and their use as sodium and/or calcium channel modulators

ABSTRACT

Substituted 2-[2-(phenyl)ethylamino]alkaneamide derivatives of formula (I) 
     
       
         
         
             
             
         
       
         
         wherein 
         X, Y, Z, R, R 1 , R 2 , R 3 , R′ 3  R 4 , R 5 , R 6 , R 7  have the meanings defined in the specification and pharmaceutically acceptable salts thereof, pharmaceutical compositions containing them as active ingredient and their use as sodium and/or calcium channel modulators useful in preventing, alleviating and curing a wide range of pathologies, including, but not limited to, neurological, cognitive, psychiatric, inflammatory, urogenital and gastrointestinal diseases, where the above mechanisms have been described as playing a pathological role.

This application is a divisional of U.S. patent application Ser. No.13/794,377, filed on Mar. 11, 2013, which is a continuation of U.S.patent application Ser. No. 12/663,926, now U.S. Pat. No. 8,519,000,filed on Apr. 30, 2010, which a U.S. national stage of PCT/EP2008/003848filed on May 14, 2008 which claims priority to and the benefit of EP07022766.8 filed Jun. 15, 2007, the contents of which are incorporatedherein by reference in their entireties.

The present invention relates to substituted2-[2-(phenyl)ethylamino]alkaneamide derivatives, pharmaceuticallyacceptable salts thereof, pharmaceutical compositions containing themand their use as sodium and/or calcium channel modulators.

The substituted 2-[2-(phenyl)ethylamino]alkaneamide derivatives of thedisclosure, are active as sodium and/or calcium channel modulators.Accordingly, they are useful in preventing alleviating and curing a widerange of pathologies, including, but not limited to neurological,cognitive, psychiatric, inflammatory, urogenital and gastrointestinaldiseases, where the above mechanisms have been described as playing apathological role.

The compounds of this invention are substantially free of monoamineoxidase (MAO) inhibitory effect, especially at dosages that aretherapeutically effective in preventing, alleviating and/or curing saidafflictions.

BACKGROUND OF THE INVENTION Chemical Background

The GB 586,645 patent describes the synthesis of amino acid derivativesof the following general formula

In particular it describes the synthesis of N-hydroxyalkylamides havingstimulating uterine smooth muscle properties.

The patent application WO 90/14334 describes mono-substitutedN-phenylalkyl alpha-amino carboxamide derivatives of the followinggeneral formula

whereinR is a (C₁-C₈)alkyl, (C₃-C₈)cycloalkyl, furyl, thienyl, pyridyl or aphenyl ring optionally substituted by 1 to 4 substituents independentlyselected from halo, (C₁-C₆)alkyl, (C₁-C₆)alkoxy and trifluoromethyl; Ais a —(CH₂)_(m)— or —(CH₂)p-X—(CH₂)q- group wherein m is an integer of 1to 4, one of p and q is zero and the other is zero or an integer of 1 to4, X is —O—, —S— or —NR₄— in which R₄ is hydrogen or (C₁-C₄)alkyl; n is0 or 1; each of R₁ and R₂ independently is hydrogen or (C₁-C₄)alkyl; R₃is hydrogen, (C₁-C₄)alkyl optionally substituted by hydroxy or phenyloptionally substituted as above; R₃′ is hydrogen or R₃ and R₃′ takentogether form a (C₃-C₆)cycloalkyl ring; each of R₅ and R₆ independentlyis hydrogen or (C₁-C₆) alkyl, with the proviso that when R is(C₁-C₈)alkyl, then A is a —(CH₂)_(p)—X—(CH₂)_(q)— group in which p and qare both zero and X is —O—, —S— or —NR₄—, in which R₄ is hydrogen orC₁-C₄ alkyl for use as anti-epileptic, anti-Parkinson, neuroprotective,anti-depressant, anti-spastic and/or hypnotic agents.

None of derivatives synthesized in our patent application isspecifically disclosed and prepared in WO 90/14334.

In other patent applications, selected compounds falling in WO 90/14334general formula, are claimed for use in compositions having otheractivities, specifically:

-   WO 99/35125 Alpha-aminoamide derivatives useful as analgesic agents-   WO 03/020273 Pharmaceutical composition comprising gabapentin or    analogue thereof and an alpha aminoamide and its analgesic use-   WO 04/062655 Alpha-aminoamide derivatives useful as antimigraine    agents-   WO 05/018627 Alpha-aminoamide derivatives useful as    anti-inflammatory agents-   WO 05/070405 Alpha-aminoamide derivatives useful in the treatment of    lower urinary tract disorders-   WO 05/102300 Alpha-aminoamide derivatives useful in the treatment of    Restless Legs Syndrome and additive disorders-   WO 06/027052 Use of (halobenzyloxy) benzylamino-propanamides for the    manufacture of medicaments active as sodium and/or calcium channel    selective modulators-   EP Appl. No 06012352.8 α-Aminoamide Derivatives Useful in the    Treatment of Cognitive Disorders.

In the patent application WO 98/35957 amide derivatives of the followinggeneral formula are described

and are claimed to be useful against obesity and eating disorders.

None of the compounds synthesized in our patent application has beenactually synthesized or specifically listed in this WO 98/35957.

-   -   In WO 2004/087125, compounds of the following general formula        are described:

A sodium channels blocking mechanism and many pharmacological activitiesare claimed, in particular anti-pain and anti bladder disfunctionactivities.

It must be underlined that when X₂ is an alkylene, it cannot be—CH₂—CH₂— but only —CH₂—, so none of the2-[2-(phenyl)ethylamino]alkaneamide derivatives of this applicationfalls within the general formula reported above.

-   -   Eleonora Ghidini et al., in Bioorganic & Medicinal Chemistry        2006, 14, 3263-3274 describe compounds of the following general        formula:

These compounds have been tested for an anticonvulsant activity.

None of the compounds disclosed and synthesized in this patentapplication falls within the general formula described above.

-   -   The co-pending application PCT/EP 2006/011443 (WO 2007/071311)        filed on Nov. 29, 2006, refers to 2-phenylethylamino derivatives        of the following general formula:

None of the compounds claimed in this application falls within thePCT/EP 2006/011443 (WO 2007/071311).

Biological Background

Sodium channels play an important role in the neuronal network bytransmitting electrical impulses rapidly throughout cells and cellnetworks, thereby coordinating higher processes ranging from locomotionto cognition. These channels are large transmembrane proteins, which areable to switch between different states to enable selective permeabilityfor sodium ions. For this process an action potential is needed todepolarize the membrane, and hence these channels are voltage-gated. Inthe past few years a much better understanding of sodium channels anddrugs interacting with them has been developed.

Voltage-gated sodium channels were originally classified based on theirsensitivity to tetrodotoxin, from low nanomolar (Tetrodotoxin sensitive,TTXs) to high micromolar (Tetrodotoxin resistant, TTXr). So far, 9different sodium channel a subunits have been identified and classifiedas Nav1.1 to Nav1.9.

Nav1.1 to Nav1.4, Nav1.6 and Nav1.7 are TTXs, whereas Nav1.5, Nav1.8 andNav.1.9 are TTXr, with different degrees of sensitivity. Nav1.1 toNav1.3 and Nav1.6, are primarily expressed in the CNS, whereas Nav1.4and Nav1.5 are mainly expressed in muscle (skeletal and heartrespectively) and Nav1.7, Nav1.8 and Nav1.9 are predominantly expressedin DRG sensory neurons.

It has become clear that a number of drugs having an unknown mechanismof action actually act by modulating sodium channel conductance,including local anaesthetics, class I antiarrhythmics andanticonvulsants. Neuronal sodium channel blockers have found applicationwith their use in the treatment of epilepsy (phenytoin andcarbamazepine), bipolar disorder (lamotrigine), preventingneurodegeneration, and in reducing neuropathic pain. Variousanti-epileptic drugs that stabilize neuronal excitability are effectivein neuropathic pain (e.g. carbamazepine).

In addition, an increase in sodium channel expression or activity hasalso been observed in several models of inflammatory pain, suggesting arole of sodium channels in inflammatory pain.

Calcium channels are membrane-spanning, multi-subunit proteins thatallow controlled entry of calcium ions into cells from the extracellularfluid. Commonly, calcium channels are voltage dependent and are referredto as voltage-gated calcium channels (VGCC). VGCCs are found throughoutthe mammalian nervous system, where they regulate the intracellularcalcium ions levels that are important for cell viability and function.Intracellular calcium ion concentrations are implicated in a number ofvital processes in animals, such as neurotransmitter release, musclecontraction, pacemaker activity and secretion of hormones. All“excitable” cells in animals, such as neurons of the central nervoussystem (CNS), peripheral nerve cells, and muscle cells, including thoseof skeletal muscles, cardiac muscles and venous and arterial smoothmuscles, have voltage dependent calcium channels.

Calcium channels are a large family with many genetically,physiologically, and pharmacologically distinct subtypes. Based on thebiophysical properties of calcium currents recorded from individualneurons, two super-families have been described: High Voltage Activated(HVA) and Low Voltage Activated (LVA) calcium channels. Calcium currentsare referred to as L-Type, P-Type, Q-Type, N-Type, and R-Type. Thisclass of calcium currents belong to the HVA super-family, whereas T-Typecurrents belong to the LVA super family. From their molecular identity,ten distinct calcium channel subtypes have been identified, cloned andexpressed and grouped into three families: the Cav1 family (Cav 1.1,1.2, 1.3, 1.4) is functionally related to the L-type Ca current; theCav2 family (Cav 2.1, 2.2, 2.3) is functionally related to the P/Q, N,R-type currents and the Cav3 (Cav 3.1, 3.2, 3.3) family is functionallyrelated to the T-type current.

It is believed that calcium channels are relevant in certain diseasestates. A number of compounds useful in treating various cardiovasculardiseases in mammals, including humans, are thought to exert theirbeneficial effects by modulating functions of voltage dependant calciumchannels present in cardiac and/or vascular smooth muscle. Compoundswith activity against calcium channels have also been implicated for thetreatment of pain. In particular N-type calcium channels (Cav2.2),responsible for the regulation of neurotransmitter release, are thoughtto play a significant role in nociceptive transmission, both due totheir tissue distribution as well as from the results of severalpharmacological studies. N-type calcium channels were found up-regulatedin the ipsilateral dorsal horn in neuropathic pain models of injury(Cizkova D., et al., Exp. Brain Res. (2002) 147: 456-463). SpecificN-type calcium channel blockers were shown to be effective in reducingpain responses in neuropathic pain models (Matthews E. A., Dickenson A.H. Pain (2001) 92: 235-246) in the phase II of the formalin test (DiazA., Dickenson A. H. Pain (1997) 69: 93-100) and the hyperalgesiainitiated by knee joint inflammation (Nebe J., Vanegas H., Schaible H.G. Exp. Brain Res. (1998) 120: 61-69). Mutant mice, lacking the N-typecalcium channels, were found to have a decreased response to persistentpain as seen by a decrease in pain response during phase II of theformalin test (Kim, et al., Mol. Cell Neurosci. (2001) 18: 235-245;Hatakeyama S., et al., Neuroreport (2001) 12: 2423-2427) as well as toneuropathic pain, assessed by a decrease in mechanical allodynia andthermal hyperalgesia in the spinal nerve ligation model. Interestingly,these mice also showed lower levels of anxiety when compared to wildtype (Saegusa H., et al., EMBO J. (2001) 20: 2349-2356). The involvementof N-type calcium channels in pain has been further validated in theclinic by ziconotide, a peptide derived from the venom of the marinesnail, Conus Magnus. A limitation in the therapeutic use of this peptideis that it has to be administered intrathecally in humans (Bowersox S.S. and Luther R. Toxicon, (1998) 36: 1651-1658).

All together these findings indicate that compounds with sodium and/orcalcium channel blockade have a high therapeutic potential inpreventing, alleviating and curing a wide range of pathologies,including neurological, psychiatric, urogenital and gastrointestinaldiseases, where the above mechanisms have been described as playing apathological role.

There are many papers and patents which describe sodium channel and/orcalcium channel modulators or antagonists for the treatment ormodulation of a plethora of disorders, such as their use as localanaesthetics, antimanic anti-depressants, agents for the treatment ofunipolar depression, urinary incontinence, diarrhoea, inflammation,epilepsy, neurodegenerative conditions, nerve cell death, neuropathicpain, migraine, acute hyperalgesia and inflammation, renal disease,allergy, asthma, bronchospasm, dysmenorrhea, esophageal spasm, glaucoma,urinary tract disorders, gastrointestinal motility disorders.

A non-exhaustive list of such papers and patents/patent applicationsdescribing sodium and/or calcium channels blockers and uses thereofincludes the references shown below:

C. Alzheimer describes in Adv. Exp. Med. Biol. 2002, 513, 161-181,sodium and calcium channels as targets of neuroprotective substances.

Vanegas e Schaible (Pain 2000, 85, 9-18) discuss effects of antagonistsof calcium channels upon spinal mechanisms of pain, hyperalgesia andallodynia.

WO 03/057219 relates to sodium channel blockers useful as agents fortreating or modulating a central nervous system disorder, such asneuropathic pain, inflammatory pain, inflammation-related pain orepilepsy.

WO99/14199 discloses substituted1,2,3,4,5,6-hexahydro-2,6-methano-3-benzazocines-10-oles as potentsodium channel blockers useful for the treatment of several diseases,such as stroke, neurodegenerative disorders, Alzheimer's Disease,Parkinson's Disease and cardiovascular disorders.

WO01/74779 discloses new aminopyridine sodium channel blockers and theiruse as anticonvulsants, local anaesthetics, as antiarrhythmics, for thetreatment or prevention of neurodegenerative conditions, such asamyotrophic lateral sclerosis (ALS), for the treatment or prevention ofboth, acute or chronic pain, and for the treatment or prevention ofdiabetic neuropathy.

WO04/087125 discloses amino acid derivatives as inhibitors of mammaliansodium channels, useful in the treatment of chronic and acute pain,tinnitus, bowel disorders, bladder dysfunction and demyelinatingdiseases.

U.S. Pat. No. 5,051,403 relates to a method of reducing neuronal damageassociated with an ischemic condition, such as stroke, by administrationof binding/inhibitory omega-conotoxin peptide wherein the peptide ischaracterized by specific inhibition of voltage-gated calcium channelcurrents selectively in neuronal tissues.

U.S. Pat. No. 5,587,454 relates to compositions and methods of producinganalgesia particularly in the treatment of pain and neuropathic pain.

U.S. Pat. No. 5,863,952 relates to calcium channel antagonists for thetreatment of ischaemic stroke.

U.S. Pat. No. 6,011,035 relates to calcium channel blockers, useful inthe treatment of conditions such as stroke and pain.

U.S. Pat. No. 6,117,841 relates to calcium channel blockers and theiruse in the treatment of stroke, cerebral ischemia, pain, head trauma orepilepsy.

U.S. Pat. No. 6,362,174 relates to N-type calcium channel blockers inthe treatment of stroke, cerebral ischemia, pain, epilepsy, and headtrauma.

U.S. Pat. No. 6,420,383 and U.S. Pat. No. 6,472,530 relate to novelcalcium channel blockers, useful for treating and preventing a number ofdisorders such as hypersensitivity, allergy, asthma, bronchospasm,dysmenorrhea, esophageal spasm, glaucoma, premature labor, urinary tractdisorders, gastrointestinal motility disorders and cardiovasculardisorders.

U.S. Pat. No. 6,458,781 relates to compounds that act to block calciumchannels and their use to treat stroke, cerebral ischemia, pain, headtrauma or epilepsy.

U.S. Pat. No. 6,521,647 relates to the use of calcium channel blockersin the treatment of renal disease in animals, especially chronic renalfailure.

WO 97/10210 relates to tricyclic heterocyclic derivatives, and their usein therapy, in particular as calcium channel antagonists, e.g. for thetreatment of ischaemia, in particular ischaemic stroke.

WO 03/018561 relates to quinoline compounds as N-type calcium channelantagonists and methods of using such compounds for the treatment orprevention of pain or nociception.

Monoamine oxidase (MAO) is an enzyme present in the outer mitochondrialmembrane of neuronal and non-neuronal cells. Two isoforms of MAO exist:MAO-A and MAO-B. MAO enzymes are responsible for the oxidativedeamination of endogenous and xenobiotic amines, and have a differentsubstrate preference, inhibitor specificity, and tissue distribution.Serotonin, noradrenaline and adrenaline are preferential substrates forMAO-A, and clorgyline is a selective MAO-A inhibitor; whereas MAO-Bprefers β-phenylethylamine as a substrate, and is inhibited byselegiline. Dopamine, tyramine and tryptamine are oxidized by both MAO-Aand MAO-B, in particular in human brain dopamine is deaminated by 80% byMAO-B.

MAO inhibition allows endogenous and exogenous substrates to accumulateand may thereby, when almost fully inhibited (>90%), alter the dynamicsof regular monoamine transmitters. MAO regulate the concentrations inthe brain of the most important neurotransmitters such as noradrenaline,serotonin and dopamine which are related to emotion, anxiety andmovement. Thus, it is thought that MAO be closely involved in variouspsychiatric and neurological disorders such as depression, anxiety andParkinson's disease (PD).

MAO-A inhibitors are mainly used in psychiatry for the treatment ofmajor, refractory and atypical depression as a consequence of theirability to increase the reduced serotonin and noradrenaline brainlevels. More recently, MAO-A inhibitors have been used to treat patientswith anxiety disorders such as social phobia, panic disorders,post-traumatic stress disorders and obsessive compulsive disorders.

MAO-B inhibitors are mainly used in neurology for the treatment of PD.

There is also recent evidence and interest in the role of MAO-B in otherpathological conditions such as Alzheimer's disease (AD). So far noevidence has been reported on MAO-B involvement in the metabolism ofco-transmitters, such as colecystokinin, substance P, somatostatin andneurotensin, which are involved in the modulation of pain sensation. Forthis reason there is no scientific rationale for the use of MAO-Binhibitors in pain syndromes. Adverse drug reactions during clinicalpractice with MAO inhibitors have been reported. The first generation ofnon-selective and irreversible MAO inhibitors, such as tranylcypromineand phenelzine, have serious side effects, including hepatotoxicity,orthostatic hypotension and, most importantly, hypertensive crisis thatoccurs following the ingestion of foods containing tyramine (Cooper AJ.—Tyramine and irreversible monoamine oxidase inhibitors in clinicalpractice.—Br J Psych Suppl 1989: 38-45).

When these non-selective and irreversible MAO inhibitors are used, astrict tyramine-reduced diet must be observed. The pressor sensitivitytowards tyramine is normalized 4 weeks after cessation oftranylcypromine therapy and more than 11 weeks after cessation ofphenelzine therapy.

Selegiline, an irreversible MAO-B inhibitor, especially when used incombination with levodopa, can cause anorexia/nausea, dry mouth,dyskinesia and orthostatic hypotension in patients with PD, the latterbeing most problematic (Volz H. P. and Gleiter C. H.—Monoamine oxidaseinhibitors. A perspective on their use in the elderly.—Drugs Aging 13(1998), pp. 341-355).

In monotherapy, anorexia/nausea, musculoskeletal injuries, and cardiacarrhythmias occurred more often in patients receiving selegilinecompared with those receiving placebo. Apart from these adverse effects,increased rates of elevated serum AST and ALT levels were noted. Themost frequently reported adverse effect of moclobemide, a selective andreversible MAO-A inhibitor, are sleep disturbances, increased anxiety,restlessness, and headache.

The combination of selective serotonin reuptake inhibitors (SSRIs) andmoclobemide has good efficacy in cases of refractory depression, but hascreated controversy as to whether toxic side effects, such asserotoninergic syndrome, result from this combination (BaumannP.—Pharmacokinetic-pharmacodynamic relationship of the selectiveserotonin reuptake inhibitors. Clin Pharmacokinet 31 (1996), pp444-469). Because of cardiac arrhythmias and increased liver enzymelevels, electrocardiogram and laboratory values should be checkedregularly.

Many types of physiologic changes that occur with aging affect thepharmacodynamics and pharmacokinetics of MAO inhibitors. Indeed,pharmacokinetic variables in the elderly are markedly different formthose in younger patients. These variables including absorption,distribution, metabolism and excretion have to be taken into account toavoid or minimize certain adverse effects and drug-drug interactions.Elderly patients are generally more susceptible than younger patients toside effects, including adverse drug reactions. Hypertensive crisis mayoccur more frequently in elderly than in younger patients, becausecardiovascular systems of the elderly are already compromised by age.

The use of sympathomimetic drugs in combination with MAO inhibitors mayalso elevate blood pressure. In addition, compared with placebo,phenelzine was associated with a significantly higher incidence ofdrowsiness, tremor, dyskinesia, diarrhea, micturition difficulties,orthostatic effects, and adverse dermatological effects. It isinteresting to note that in the elderly, headache is reported with ahigher frequency than in younger patients during treatment withmoclobemide (Volz H. P. and Gleiter C. H.—Monoamine oxidase inhibitors.A perspective on their use in the elderly. Drugs Aging 13 (1998), pp.341-355).

MAO inhibitors (preferentially MAO-A, but also non selectiveMAO-A/MAO-B) are sometimes prescribed for depression. Because of thepotential risk of suicide, adverse drug reactions and toxicity due tooverdose are important factors to consider when choosing anantidepressant. In addition, when MAO inhibitors are used in highdosage, adverse cardiovascular effects seem to increase considerably;and because MAO selectivity is lost with such high doses, tyramine caninduce potentially dangerous hypertensive reactions. Acute overdose withMAO inhibitors causes agitation, hallucinations, hyperpyrexia,hyperreflexia and convulsions. Abnormal blood pressure is also a toxicsign, so that gastric lavage and maintenance of cardiopulmonary functionmay be required. Overdose of traditional non-selective and irreversibleMAO inhibitors are considerably dangerous and sometimes fatal (Yamadaand Richelson, 1996. Pharmacology of antidepressants in the elderly. In:David J R, Snyder L., editors. Handbook of pharmacology of aging. BocaRaton: CRC Press 1996).

In the treatment of the afflictions wherein sodium and calcium channelsmechanism(s) play(s) a pathological role, the inhibition of MAO enzymesis of no benefit. Moreover MAO inhibitory side effects may impose atleast two types of negative limitations:

1) Dietary: eating food with high tyramine content may cause severe,even life threatening increase of systemic blood pressure (the so called“cheese-effect”).

2) Pharmacological: as an example, pain is often treated with acombination of drugs such as opioid derivatives and tricyclicantidepressant. With MAO inhibitors such association is dangerous as itmay cause the serotoninergic syndrome (agitation, tremors,hallucination, hyperthermia and arrhythmias).

Thus, eliminating or significantly reducing MAO inhibitory activity inmedicaments active as sodium and/or calcium channel modulators useful inpreventing, alleviating and curing a wide range of pathologies wheresaid mechanism(s) play(s) a pathological role, including neurological,psychiatric, inflammatory, urogenital and gastrointestinal diseases, isan unexpected and substantial therapeutic improvement versus compoundsof similar efficacy but with the above mentioned side effects.

Taking into account these findings on MAO inhibitors and, in particular,in view of the lack of evidence on the role of MAO-B in pathologicalafflictions like pain, migraine, inflammatory, urogenital andgastrointestinal diseases, MAO-B inhibition should not be an essentialfeature for compounds indicated for the above pathologies. Suchcompounds would thereby avoid producing possible side effects duringchronic and/or long-term treatments.

An advantageous solution to the above described problem would consist inproviding medicaments which are “selectively active as sodium and/orcalcium modulators” or are useful for the “selective treatment” ofafflictions, disorders or diseases wherein the sodium and/or calciumchannel mechanism(s) play(s) a pathological role. Selective treatmentand selective activity as sodium and/or calcium modulators means thatthe medicaments when administered to a patient in need thereof inamounts that are effective in the treatment of the above saidafflictions wherein the above said mechanism(s) play(s) pathologicalrole do not exhibit any MAO inhibitory activity or exhibit asignificantly reduced MAO inhibitory activity, thereby avoiding of sideeffects due to accumulation of endogenous and exogenous monoaminetransmitters.

One primary object of this invention is the use of2-[2-(phenyl)ethylamino]alkaneamide derivatives for the manufacture of amedicament active as sodium and/or calcium channel modulator for thetreatment of pathologies where the above said mechanism(s) play(s) apathological role, which is a neurological, cognitive, psychiatric,inflammatory, urogenital or gastrointestinal disorder, said medicamentsbeing substantially free from any MAO inhibitory activity or havingsignificantly reduced MAO inhibitory activity and, therefore, having areduced potential for unwanted side effects. Accordingly, a main objectof this invention is to provide 2-[2-(phenyl)ethylamino]alkaneamidederivatives for use as medicaments for treating the above describedpathologies, characterized in that said medicaments are substantiallyfree from any MAO inhibitory activity or present a significantly reducedMAO inhibitory activity and, therefore, have a reduced potential forunwanted side effects. Said use provides an improved selective resourcefor the prevention, alleviation and/or cure of the above saidpathological afflictions, in particular, in patients who areparticularly sensitive to unwanted side-effects due to MAO inhibitoryactivity, such as those hereinabove described.

A further aspect of this invention is to provide a method for treating apatient affected by a disorder caused by dysfunctions of voltage-gatedsodium and/or calcium channels which comprises the administration tosaid patient of an effective amount of a2-[2-(phenyl)ethylamino]alkaneamide derivative.

The above mentioned neurological disorders include pain of both chronicand acute type, in particular neuropathic and inflammatory pain,headaches, migraine, spasms; neurodegenerative disorders such asAlzheimer's disease, Parkinson's disease, epilepsy, restless legssyndrome, stroke and cerebral ischemia; cognitive disorders such as MildCognitive Impairment (MCI) and psychiatric disorders includingdepression, bipolar disorders, mania, schizophrenia, psychoses, anxietyand addiction. The above mentioned inflammatory disorders includeinflammatory processes affecting all body systems, e.g. inflammatoryprocesses of the muscle-skeletal system, arthritic conditions, disordersaffecting skin and related tissues; disorders of the respiratory systemas well as disorders of immune and endocrinological system. A moredetailed explanation of all above mentioned pathologies is givenhereinafter in the following.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides results from testing of a rat spinal nerve ligationmodel of neuropathic pain further described in Example 17;

FIG. 2 provides results from testing of a mouse acetic acid-inducedmodel of visceral pain, further described in Example 19;

FIG. 3 provides results for antimania activity as measured using a mousemodel having amphetamine and chlordiazepoxide-induced hyperlocomotion,further described in Example 21; and

FIG. 4 provides results for a test of prepulse inhibition of startle(PPI) in mice and in rats, further described in Example 26.

DESCRIPTION OF THE INVENTION

The object of this application is a new class of2-[2-(phenyl)ethylamino]alkaneamide derivatives highly potent as sodiumand/or calcium channel modulators and substantially free from any MAOinhibitory activity or having significantly reduced MAO inhibitoryactivity and, thus, having potentially reduced side effects inpreventing, alleviating and curing a wide range of pathologies,including but not limited to neurological, cognitive, psychiatric,inflammatory, urogenital and gastrointestinal diseases where the abovemechanisms have been described as playing a pathological role.

In this description and claims, the expression “sodium and/or calciumchannel modulator(s)” means compounds able to block sodium and/orcalcium currents in a voltage dependent manner.

Therefore, object of the present invention is a compound of generalformula (I)

wherein:

-   X is —O—, —S— or —SO₂—;-   Y is hydrogen, OH or O(C₁-C₄)alkyl;-   Z is ═O or ═S;-   R is (C₃-C₁₀)alkyl; ω-trifluoro(C₃-C₁₀)alkyl;-   R₁ and R₂ are, independently, hydrogen, hydroxy, (C₁-C₈)alkoxy,    (C₁-C₈) alkylthio, halo, trifluoromethyl or 2,2,2-trifluoroethyl; or    one of R₁ and R₂ is in ortho position to R—X— and, taken together    with the same R—X—, represents a

group where R₀ is (C₂-C₉)alkyl;

-   R₃ and R′₃ are, independently, hydrogen or (C₁-C₄)alkyl;-   R₄ and R₅ are, independently, hydrogen, (C₁-C₄)alkyl; or R₄ is    hydrogen and R₅ is a group selected from —CH₂—OH,    —CH₂—O—(C₁-C₆)alkyl, —CH(CH₃)—OH, —(CH₂)₂—S—CH₃, benzyl and    4-hydroxybenzyl; or R₄ and R₅, taken together with the adjacent    carbon atom, form a (C₃-C₆)cycloalkyl residue;-   R₆ and R₇ are independently hydrogen or (C₁-C₆)alkyl; or taken    together with the adjacent nitrogen atom form a 5-6 membered    monocyclic saturated heterocycle, optionally containing one    additional heteroatom chosen among —O—, —S— and —NR₈— where R₈ is    hydrogen or (C₁-C₆) alkyl;    with the proviso that when X is —S— or —SO₂—, then Y is not OH or    O(C₁-C₄) alkyl;    if the case, either as single optical isomer in the isolated form or    mixture thereof in any proportion and its pharmaceutically    acceptable salts.

Although some of the compounds specifically described in thisapplication are falling within the general formula of WO 90/14334, noneof them has been specifically described in said application. None of thecompounds defined by the general formula (I) of this application isspecifically described or mentioned in WO 90/14334. In fact only few2-(2-phenyl-ethylamino)alkaneamide derivatives are identified in saidprior document which, however, have a benzyloxy, benzylamino or benzylsubstituent in the 4-position of the phenyl portion. Moreover, thefollowing selected classes of compounds of formula (I) of thisapplication do not fall within the general formula of WO 90/14334:

-   -   a) compounds where X is —SO₂—;    -   b) compounds where Y is OH or O(C₁-C₄)alkyl;    -   c) compounds where Z is ═S;    -   d) compounds where R is (C₉-C₁₀)alkyl or        ω-trifluoro(C₃-C₁₀)alkyl;    -   e) compounds where R₁ and/or R₂ are different from hydrogen;    -   f) compounds where both R₃ and R′₃ are different from hydrogen    -   g) compounds where both R₄ and R₅ are different from hydrogen,        but do not form a (C₃-C₆)cycloalkyl residue when taken together        with the adjacent carbon atom;    -   h) compounds where R₆ and R₇, taken together with the adjacent        nitrogen atom form a monocyclic 5-6 membered saturated        heterocycle, optionally containing one additional heteroatom        chosen among —O—, —S— and —NR₈—, where R₈ is hydrogen or (C₁-C₆)        alkyl.

The term “alkyl” used in this description and claims, where no otherwisespecified, identifies a straight or branched alkyl radical; examples ofsaid radicals or moieties include: methyl, ethyl, propyl, isopropyl,butyl, isobutyl, pentyl, isopentyl, hexyl, heptyl, octyl, nonyl, decyland their isomers.

The term “alkoxy” used in this description and claims identifies astraight or branched alkoxy radical; examples of said radicals include:methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy,pentyloxy, isopentyloxy, hexyloxy, isohexyloxy, heptyloxy, octyloxy andtheir isomers.

The term “(C₃-C₆)cycloalkyl” identifies a cycloaliphatic ring; examplesof said rings include: cyclopropyl, cyclobutyl, cyclopentyl andcyclohexyl.

The term “halo” means an halogen atom radical such as fluoro, chloro,bromo and iodo. Examples of a monocyclic 5 or 6 membered saturatedheterocycles, optionally containing one additional heteroatom, chosenamong —O—, —S— or —NR₈—, where R₈ is hydrogen or (C₁-C₆) alkyl are, forinstance, pyrrolidine, pyrazolidine, imidazolidine, oxazolidine,isoxazolidine, piperidine, piperazine, N-methylpiperazine, morpholineand thiomorpholine. When the compounds of this invention contain one ormore asymmetric carbon atoms and, therefore, they can exist as singleoptical isomers or a mixture thereof, the invention includes within itsscope all the possible single optical isomers, (e.g. enantiomers,diastereoisomers) of said compounds in the isolated form and themixtures thereof in any proportion, including racemic mixtures.

Examples of pharmaceutically acceptable salts of the compounds offormula (I) are salts with organic and inorganic acids such ashydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric,acetic, propionic, tartaric, fumaric, citric, benzoic, succinic,cinnamic, mandelic, salicylic, glycolic, lactic, oxalic, malic, maleic,malonic, fumaric, tartaric, p-toluenesulfonic, methanesulfonic, glutaricacid and other acids which, for instance, can be found in: P. HeinrichStahl, Camille G. Wermuth “Handbook of pharmaceutical salts: properties,selection and use”, WILEY-VCH, 2002.

The compounds of formula (I) are active as sodium and/or calcium channelmodulators and therefore, are useful in preventing, alleviating andcuring a wide range of pathologies, including but not limited toneurological, cognitive, psychiatric, inflammatory, urologic, andgastrointestinal diseases, where the above mechanisms have beendescribed as playing a pathological role.

Preferred compounds of formula (I) are the compounds wherein:

-   X is —O—, —S—;-   Y is hydrogen, OH or O(C₁-C₃)alkyl;-   Z is ═O or ═S;-   R₁ and R₂ are, independently, hydrogen, (C₁-C₄)alkoxy, halo,    trifluoromethyl or 2,2,2-trifluoroethyl; or one of R₁ and R₂ is in    ortho position to R—X— and, taken together with the same R—X—,    represent a

group where R₀ is (C₂-C₅)alkyl;

-   R₃ and R′₃ are, independently, hydrogen or (C₁-C₃)alkyl;-   R₄ and R₅ are, independently, hydrogen or (C₁-C₄)alkyl; or R₄ is    hydrogen and R₅ is a group selected from —CH₂—OH,    —CH₂—O—(C₁-C₃)alkyl, —(CH₂)₂—S—CH₃, benzyl and 4-hydroxybenzyl;-   R₆ and R₇ are, independently, hydrogen or (C₁-C₄)alkyl; or taken    together with the adjacent nitrogen atom form a 5-6 membered    monocyclic saturated heterocycle, optionally containing one    additional heteroatom chosen among —O— and —NR₈— where R₈ is    hydrogen or (C₁-C₃) alkyl;    with the proviso that when X is —S—, then Y is not OH or O(C₁-C₄)    alkyl    if the case, either as single optical isomers in the isolated form    or mixture thereof in any proportion and their pharmaceutically    acceptable salts.

More preferred compounds of formula (I) are the compounds wherein:

-   X is —O—, —S—;-   Y is hydrogen or O(C₁-C₃)alkyl;-   Z is ═O or ═S;-   R is (C₄-C₇)alkyl or ω-trifluoro(C₄-C₆)alkyl;-   R₁ and R₂ are, independently, hydrogen, (C₁-C₃)alkoxy, fluoro,    chloro, trifluoromethyl or 2,2,2-trifluoroethyl; or one of R₁ and R₂    is in ortho position to R—X— and taken together with the same R—X—,    represents a

group where R₀ is (C₃-C₄)alkyl;

-   R₃ and R′₃ are, independently, hydrogen or (C₁-C₃)alkyl;-   R₄ and R₅ are, independently, hydrogen or (C₁-C₄)alkyl; or R₄ is    hydrogen and R₅ is a group selected from —CH₂—OH,    —CH₂—O—(C₁-C₃)alkyl, benzyl and 4-hydroxybenzyl;-   R₆ and R₇ are, independently, hydrogen or (C₁-C₃)alkyl; or taken    together with the adjacent nitrogen atom form a 5-6 membered    monocyclic saturated heterocycle, optionally containing one    additional heteroatom chosen among —O— and —NR₈—, where R₈ is    hydrogen or (C₁-C₃) alkyl;    with the proviso that when X is —S—, then Y is not O(C₁-C₄) alkyl;    if the case, either as single optical isomers in the isolated form    or mixture thereof in any proportion and their pharmaceutically    acceptable salts.

Even more preferred compounds of formula (I) are those compoundswherein:

-   X is —O—;-   Y is hydrogen;-   Z is ═O;-   R is (C₄-C₆)alkyl;-   R₁ and R₂ are, independently, hydrogen or halo, preferably fluoro;-   R₃, R′₃, R₄ and R₅ are hydrogen;-   R₆ and R₇ are, independently, hydrogen or (C₁-C₃)alkyl;    if the case, either as single optical isomers in the isolated form    or mixture thereof in any proportion and their pharmaceutically    acceptable salts;

Most preferably, a compound of formula (I) according to this inventionis selected from the group consisting of:

-   2-[2-(3-Butoxyphenyl)-ethylamino]-acetamide-   2-[2-(3-Pentyloxyphenyl)-ethylamino]-acetamide-   2-[2-(3-Hexyloxyphenyl)-ethylamino]-acetamide-   2-[2-(3-Butoxyphenyl)-ethylamino]-N-methylacetamide-   2-[2-(3-Pentyloxyphenyl)-ethylamino]-N-methylacetamide-   2-[2-(3-Hexyloxyphenyl)-ethylamino]-N-methylacetamide-   2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butylthiophenyl)-ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butylsulfonylphenyl)-ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butoxyphenyl)-(N′-hydroxy)ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butoxyphenyl)-(N′-methoxy)ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butoxyphenyl)-(N′-propoxy)ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dimethyl-thioacetamide-   2-[2-(3-Butoxyphenyl)-2-methylpropylamino]-N,N-dimethylacetamide-   2-{2-[3-(4,4,4-Trifluorobutoxy)phenyl]-ethylamino}-N,N-dimethylacetamide-   2-[2-(3-Butylthiophenyl)-ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butoxy-2-chlorophenyl)-ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butoxy-2-fluorophenyl)-ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butoxy-4-methoxyphenyl)-ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butoxy-4-methylphenyl)-ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butoxy-2,4-difluoro-4-methylphenyl)-ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butoxy-2,6-difluoro-4-methylphenyl)-ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-diethylacetamide-   2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dipropylacetamide-   2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dibutylacetamide-   2-[2-(3-Pentyloxyphenyl)-ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Hexyloxyphenyl)-ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butoxyphenyl)-ethylamino]-1-pyrrolidin-1-yl-ethan-1-one-   2-[2-(3-Pentyloxyphenyl)-ethylamino]-1-pyrrolidin-1-yl-ethan-1-one-   2-[2-(3-Hexyloxyphenyl)-ethylamino]-1-pyrrolidin-1-yl-ethan-1-one-   2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dimethylpropanamide-   2-[2-(3-Butoxyphenyl)-ethylamino]-3-hydroxy-N,N-dimethylpropanamide-   2-[2-(3-Butoxyphenyl)-ethylamino]-3-methoxy-N,N-dimethylpropanamide-   2-[2-(3-Butoxyphenyl)-ethylamino]-3-propoxy-N,N-dimethylpropanamide-   2-[2-(3-Butoxyphenyl)-ethylamino]-2,N,N-trimethylpropanamide-   2-[2-(3-Pentyloxyphenyl)-ethylamino]-2,N,N-trimethylpropanamide-   2-[2-(3-Hexyloxyphenyl)-ethylamino]-2,N,N-trimethylpropanamide-   (S)-2-[2-(3-Butoxyphenyl)-ethylamino]-propanamide-   (S)-2-[2-(3-Butoxyphenyl)-ethylamino]-N-methylpropanamide-   (S)-2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dimethylpropanamide-   (R)-2-[2-(3-Butoxyphenyl)-ethylamino]-propanamide-   (R)-2-[2-(3-Butoxyphenyl)-ethylamino]-N-methylpropanamide-   (R)-2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dimethylpropanamide-   2-[2-(3-Butoxy-2-trifluoromethylphenyl)-ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butoxy-4-trifluoromethylphenyl)-ethylamino]-N,N-dimethylacetamide-   2-[2-(3-Butoxy-5-trifluoromethylphenyl)-ethylamino]-N,N-dimethylacetamide    if the case, either as single optical isomers in the isolated form    or mixtures thereof in any proportion and their pharmaceutically    acceptable salts, preferably their salts with hydrochloric or    methanesulfonic acid.

The compounds of this invention are prepared according to conventionalprocedures which are described in more detail in the Experimental Part.

In particular most of the compounds of formula (I), where X is —O— and Yis hydrogen, object of the present invention, are prepared according toa synthetic process shown in the following

Scheme I:

wherein:R, R₁, R₂, R₃, R′₃, R₄, R₅, R₆ and R₇ have the same meanings defined informula (I) above, Ph means a phenyl radical, boc is atert-butoxycarbonyl group and EWG stays for “Electron Withdrawing Group”such as, for example, a halogen or a mesyloxy or a tosyloxy or atrifluoromethanesulfonate group. Lawessons's reagent is2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide (TheMerck Index, 13^(th) Ed., 5408, page 966).

According to a preferred embodiment of the invention the alkylationreactions with R-EWG are carried out in the presence of a base and, morepreferably, said base is selected from K₂CO₃, triethylamine anddiisopropylethylamine.

An alternative method for the preparation of the compounds of formula(I) where R, R₁, R₂, R₃, R′₃, R₄, R₅, R₆ and R₇ have the same meaningsas in formula (I), X is O and Y is hydrogen consists in submitting analdehyde of the formula

to a reductive alkylation with an α-aminoalkaneamide of formula

The reducing agent may be selected from NaBH₄, NaBH₃CN and(polystyrylmethyl)-trimethylammonium cyanoborohydride.

The resulting 2-[2-(phenyl)ethylamino]alkaneamide compounds of formula(I) wherein Z is ═O can be transformed into the corresponding compoundswhere Z is ═S by protecting the —NH— group with boc₂O, reacting theN-boc protected derivative with Lawesson's reagent, and finallydeprotecting with HCl as show in Scheme I.

As a further alternative to these methods, an amine of formula

is alkylated by reaction with an ester of an alkanoic acid of formula

in the presence of a base (e.g. triethylamine) and the resultingalkanoic ester derivatives of formula

is reacted with an amine of formula HNR₆R₇, optionally in the presenceof an amidation catalyst (e.g. trimethylaluminium), to yield thecompound formula (I) where Y is hydrogen. The compounds of formula (I)where R₅ is hydrogen, optionally can be transformed into thecorresponding compounds of formula (I) wherein R₅ is (C₁-C₄)alkyl,—CH₂OH, —CH₂—O—(C₁-C₆) alkyl, —CH(CH₃)—OH, —(CH₂)₂—S—CH₃, benzyl, or4-hydroxybenzyl by submitting a N-protected derivative of the above saidcompound of formula (I) to a C-alkylation procedure with an alkylatingagent of formula EWGR₅ wherein EWG has the same meaning as above and R₅represents one of the group listed hereinabove. In this case, when a endcompound of formula (I) is desired where the group R₅ contain a hydroxymoiety, a reagent EWGR₅ is usually employed where the correspondinghydroxy moiety is protected, e.g. by acetylation. All protecting groupsare then removed from the resulting C-alkylated compounds.

If desired, when a compound of formula (I) is obtained in a form of afree base it may be converted into a salt thereof (e.g., withhydrochloric acid) by common procedures.

The compounds of formula (I), where X is S or SO₂ and Y is hydrogen areprepared according to a synthetic process shown in the following SchemeII:

whereinR, R₁, R₂, R₃, R′₃, R₄, R₅, R₆ and R₇ have the same meanings as definedin formula (I), boc is a tert-butoxycarbonyl group and EWG has the samemeaning as above, “oxone” stays for potassium peroxymonosulfate andLawessons's reagent is2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide.

The compounds of formula (I), where X is —O— and Y is OH orO(C₁-C₄)alkyl, are prepared according to a synthetic process shown inthe following Scheme III:

whereinR, R₁, R₂, R₃, R′₃, R₄, R₅, R₆ and R₇ have the same meanings as definedin formula (I), Ph means a phenyl group, and EWG has the same meaning asabove. Dress-Martin periodinane, see: Dess, D. B.; Martin, J. C. J. Am.Chem. Soc., 1991, 113, 7277.

The intermediates used in Schemes I, II and III are commerciallyavailable or are prepared from commercially available compoundsaccording to well-known methods.

The evaluation of the usefulness of the optional protection as well asthe selection of the suitable protecting agent, according to thereaction carried out in the preparation of the compounds of theinvention and the functional group to be protected, are within thecommon knowledge of the skilled person.

The removal of the optional protective groups is carried out accordingto conventional techniques.

For a general reference to the use of protective groups in organicchemistry see Theodora W. Greene and Peter G. M. Wuts “Protective groupsin organic synthesis”, John Wiley & Sons, Inc., II Ed., 1991.

The preparation of the salts of the compounds of formula (I) is carriedout according to known methods.

For the preparation of a single optical isomer of a compound of formula(I) said compound may be obtained through a sterically controlledsynthesis or by using reagents having the appropriate chirality orseparating the desired isomer from the enantiomeric mixture thereof,according to conventional procedures.

Pharmacology

The compounds of the invention may be used for the manufacture of amedicament active as sodium and/or calcium channel modulator againstdisorders caused by dysfunctions of voltage gated sodium and/or calciumchannels.

Such compounds are voltage-dependent blockers of the sodium and/orcalcium channels with potency in the low micromolar range asdemonstrated by the blockade of the sodium and/or calcium influx(fluorescence assays) and by the voltage-dependent blockade of thecurrents (patch clamp techniques).

The activity of the compounds representative of this invention wascompared with that of compounds known from WO 90/14334, which have beenclinically developed for therapeutical applications such as“ralfinamide” (S)-(+)-2-[4-(2-fluorobenzyloxy)-benzylamino]-propanamideand/or “safinamide”(S)-(+)-2-[4-(3-fluorobenzyloxy)-benzylamino]-propanamide.

Safinamide (NW-1015, FCE-26743A, PNU-151774E) is a sodium channelblocker, a calcium channel modulator, a monoamino oxidase B (MAO-B)inhibitor, a glutamate release inhibitor and a dopamine metabolismmodulator.

Safinamide is useful in the treatment of CNS disorders, in particular ofepilepsy, Parkinson's disease, Alzheimer's disease, restless legssyndrome (WO 90/14334, WO 04/089353, WO 05/102300) and cognitivedisorders (EP Appl. No 06/012352.8).

Ralfinamide (NW-1029, FCE-26742A, PNU-0154339E) is a sodium and calciumchannel inhibitor and NMDA receptor modulator useful in the treatment ofpain conditions, including neuropathic and inflammatory pain of bothacute and chronic type, migraine, depressions, cardiovascular,inflammatory, urogenital, metabolic and gastrointestinal disorders (WO99/35125, WO 03/020273, WO 04/062655, WO 05/018627, WO 05/070405, WO06/027052).

The sodium channel modulating activity of the2-[2-(phenyl)ethylamino]alkaneamide derivatives was measured through afluorescence-based sodium influx assay in ND7/23 cell line (Table 1) andthrough the electrophysiological patch clamp technique in rat corticalneurons (Table 3) and in ND7/23 cell line (Table 4) respectively.

The calcium channel modulating activity of the2-[2-(phenyl)ethylamino]alkaneamide derivatives was measured through afluorescence-based calcium influx assays (Table 2) in AtT20 cell line.

The MAO-B activity of the above compounds was measured by using aradioenzymatic assay (Table 5) in rat brain mitochondria.

The in vivo analgesic activity of the above compounds was assessed inthe “formalin test” in mice (Table 6) and in the “Spinal nerve ligationmodel of neuropathic pain (SNL)” in rats (FIG. 1). The anti-inflammatorypain activity was measured using the “Complete Freund's adjuvant model(CFA)” in rats.

The anti-visceral pain activity was measured using the “aceticacid-induced visceral pain” model in mice (FIG. 2).

The anticonvulsant activity was measured using the “Maximal electroshocktest” in mice (Table 7 and Table 8).

The anti mania activity was measured using the “Amphetamine andchlordiazepoxide-induced hyperlocomotion in mice” model (FIG. 3) and inthe “paradoxical sleep deprivation” rat model.

The antiamnesic activity was assessed using the “Morris Water Maze test”in which amnesia is induced by scopolamine in rats and in the “NovelObject Recognition test” in rats.

To investigate the antidepressant activity of the compounds the “Tailsuspension test” model in mice was used.

The anti-schizophrenia activity was assessed using the “test ofcognitive impairment in schizophrenia” and the “Prepulse inhibition ofstartle (PPI)” in mice and in rats (FIG. 4).

The “Cocaine-induced behavioural sensitization test” in rats was used toassess the anti-addiction activities of the compounds.

“Acute bladder irritation by acetic acid in rats” and “Intermediatebladder irritation by cyclophosphamide in rats” tests were used asmodels for urological diseases.

The anti migraine activity was measured using the “migraine test” inrats.

In electrophysiological patch clamp studies, such substances exhibitalso “use and frequency-dependency”, i.e. an enhancement of the blockduring a high frequency stimulation when there is a large accumulationof channels in the inactivated state, such as in neuronal pathologicalconditions. Functionally, the use-dependent block results in depressionof neuronal activity at high frequency firing and with lower blockingcapacity at normal firing rate suggesting that the compounds of thisinvention may selectively depress abnormal activity of the sodium and/orcalcium channels, leaving unaffected the physiological activity, thushaving limited CNS depressant effects (W. A. Catterall, TrendsPharmacol. Sci. (1987) 8: 57-65).

One of the most representative compounds of this invention is2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dimethylacetamide hydrochloride(NW-3509) that was found very active in almost all experimental in vitroand in vivo models.

The compounds of the invention are active in vivo when orally,intraperitoneally or intravenously administered in the range of 0.1 to100 mg/kg in different animal models here following described.

In view of the above described mechanisms of action, the compounds ofthe present invention are useful in the prevention or treatment ofneuropathic pain. Neuropathic pain syndromes include, and are notlimited to: diabetic neuropathy; sciatica; non-specific lower back pain;multiple sclerosis pain; fibromyalgia; HIV-related neuropathy;neuralgia, such as post-herpetic neuralgia and trigeminal neuralgia,Morton's neuralgia, causalgia; and pain resulting from physical trauma,amputation, phantom limb, cancer, toxins or chronic inflammatoryconditions; central pain such as the one observed in thalamic syndromes,mixed central and peripheral forms of pain such as complex regional painsyndromes (CRPS) also called reflex sympathetic dystrophies.

The compounds of the invention are also useful for the treatment ofchronic pain. Chronic pain includes, and is not limited to, chronic paincaused by inflammation or an inflammatory-related condition,osteoarthritis, rheumatoid arthritis, acute injury or trauma, upper backpain or lower back pain (resulting from systematic, regional or primaryspine disease such as radiculopathy), bone pain (due to osteoarthritis,osteoporosis, bone metastasis or unknown reasons), pelvic pain, spinalcord injury-associated pain, cardiac chest pain, non-cardiac chest pain,central post-stroke pain, myofascial pain, sickle cell pain, cancerpain, Fabry's disease, AIDS pain, geriatric pain or pain caused byheadache, temporomandibular joint syndrome, gout, fibrosis or thoracicoutlet syndromes, in particular rheumatoid arthritis and osteoarthritis.

The compounds of the invention are also useful in the treatment of acutepain (caused by acute injury, illness, sports-medicine injuries, carpaltunnel syndrome, burns, musculoskeletal sprains and strains,musculotendinous strain, cervicobrachial pain syndromes, dyspepsis,gastric ulcer, duodenal ulcer, dysmenorrhoea, endometriosis or surgery(such as open heart or bypass surgery), post operative pain, kidneystone pain, gallbladder pain, gallstone pain, obstetric pain or dentalpain.

The compounds of the invention are also useful in the treatment ofheadaches, migraine, tension type headache, transformed migraine orevolutive headache, cluster headache, as well as secondary headachedisorders, such as the ones derived from infections, metabolic disordersor other systemic illnesses and other acute headaches, paroxysmalhemicrania and the like, resulting from a worsening of the abovementioned primary and secondary headaches.

The compounds of the invention are also useful for the treatment ofneurological conditions such as epilepsy including simple partialseizure, complex partial seizure, secondary generalized seizure, furtherincluding absence seizure, myoclonic seizure, clonic seizure, tonicseizure, tonic clonic seizure and atonic seizure. The compounds of theinvention are also useful for the treatment of neurodegenerativedisorders of various origins including Alzheimer Disease, ParkinsonDisease and other dementia conditions such as Lewys body,fronto-temporal dementia and taupathies; amyotrophic lateral sclerosisand other parkinsonian syndromes; other spino cerebellar degenerationand Charcot-Marie-Toot neuropathy, traumatic brain injury, stroke andcerebral ischemia.

The compounds of the invention are also useful for the treatment ofcognitive disorders and of psychiatric disorders. Examples of cognitivedisorders are Mild Cognitive Impairment (MCI) and those associated toautism, dyslexia, attention deficit hyperactivity disorders,schizophrenia, obsessive compulsive disorders, psychosis, bipolardisorders, depression, Tourette's syndrome, and disorders of learning inchildren, adolescent and adults, Age Associated Memory Impairment, AgeAssociated Cognitive Decline, Alzheimer's Disease, Parkinson's Disease,Down's Syndrome, traumatic brain injury, Huntington's Disease,Progressive Supranuclear Palsy (PSP), HIV, stroke, vascular diseases,Pick's or Creutzfeldt-Jacob disease, multiple sclerosis (MS) and otherwhite matter disorders and drug-induced cognitive worsening. Psychiatricdisorders include, and are not limited to major depression, apathy,dysthymia, mania, bipolar disorder (such as bipolar disorder type I,bipolar disorder type II), cyclothymic disorder, rapid cycling,ultradian cycling, mania, hypomania, schizophrenia, schizophreniformdisorders, schizoaffective disorders, personality disorders, attentiondisorders with or without hyperactive behaviour, delusional disorders,brief psychotic disorders, shared psychotic disorders, psychoticdisorder due to a general medical condition, substance-induced psychoticdisorders or a psychotic disorder not otherwise specified, anxietydisorders such as generalised anxiety disorder, panic disorders,post-traumatic stress disorder, impulse control disorders, phobicdisorders, dissociative states and moreover in smoke and drug addictionand alcoholism. In particular bipolar disorders, schizophrenia,psychosis, anxiety and addiction.

The compounds of the invention inhibit inflammatory processes affectingall body systems. Therefore are useful in the treatment of inflammatoryprocesses of the muscular-skeletal system of which the following is alist of examples but it is not comprehensive of all target disorders:arthritic conditions such as alkylosing spondylitis, cervical arthritis,fibromyalgia, gut, juvenile rheumatoid arthritis, lumbosacral arthritis,osteoarthritis, osteoporosis, psoriatic arthritis, rheumatic disease;disorders affecting skin and related tissues: eczema, psoriasis,dermatitis and inflammatory conditions such as sunburn; disorders of therespiratory system: asthma, allergic rhinitis and respiratory distresssyndrome, lung disorders in which inflammation is involved such asasthma and bronchitis; chronic obstructive pulmonary disease; disordersof the immune and endocrinological systems: periarthritis nodosa,thyroiditis, aplastic anaemia, sclerodoma, myasthenia gravis, multiplesclerosis and other demyelinizating disorders, encephalomyelitis,sarcoidosis, nephritic syndrome, Bechet's syndrome, polymyositis,gingivitis.

Compounds of the invention are also useful in the treatment ofgastrointestinal (GI) tract disorders such as inflammatory boweldisorders including but not limited to ulcerative colitis, Crohn'sdisease, ileitis, proctitis, celiac disease, enteropathies, microscopicor collagenous colitis, eosinophilic gastroenteritis, or pouchitisresulting after proctocolectomy and post ileonatal anastomosis, andirritable bowel syndrome including any disorders associated withabdominal pain and/or abdominal discomfort such as pylorospasm, nervousindigestion, spastic colon, spastic colitis, spastic bowel, intestinalneurosis, functional colitis, mucous colitis, laxative colitis andfunctional dyspepsia; but also for treatment of atrophic gastritis,gastritis varialoforme, ulcerative colitis, peptic ulceration, pyresis,and other damage to the GI tract, for example, by Helicobacter pylori,gastroesophageal reflux disease, gastroparesis, such as diabeticgastroparesis; and other functional bowel disorders, such asnon-ulcerative dyspepsia (NUD); emesis, diarrhoea, and visceralinflammation.

Compounds of the invention are also useful in the treatment of disordersof the genito-urinary tract such as overactive bladder, prostatitis(chronic bacterial and chronic non-bacterial prostatitis), prostadynia,interstitial cystitis, urinary incontinence and benign prostatichyperplasia, annexities, pelvic inflammation, bartholinities andvaginitis. In particular overactive bladder and urinary incontinence.

It will be appreciated that the compounds of the invention mayadvantageously be used in conjunction with one or more other therapeuticagents. Examples of suitable agents for adjunctive therapy include aserotonin receptor modulator including a 5HT1B/1D agonist, such as atriptan (e.g. sumatriptan or naratriptan); an adenosine A1 agonist; anadenosine A2 antagonist; a purinergic P2X antagonist, an EP ligand; anNMDA modulator, such as a glycine antagonist; an AMPA modulator; asubstance P antagonist (e.g. an NK1 antagonist); a cannabinoid; anicotinic receptor agonist; an alpha-1 or 2 adrenergic agonist;acetaminophen or phenacetin; a 5-lipoxygenase inhibitor; a leukotrienereceptor antagonist; a DMARD (e.g. methotrexate); gabapentin, pregabalinand related compounds; L-dopa and/or dopamine agonists; acatechol-O-methyltransferase inhibitor; a tricyclic antidepressant (e.g.amitryptiline); a neurone stabilising antiepileptic drugs; amonoaminergic uptake inhibitor (e.g. venlafaxine); a matrixmetalloproteinase inhibitor; a nitric oxide synthase (NOS) inhibitor,such as an iNOS or an nNOS inhibitor; a free radical scavenger; analpha-synuclein aggregation inhibitor; a cholinesterase inhibitor, acholesterol lowering agent; an alpha-secretase modulator; abeta-secretase modulator; a beta-amyloid aggregation inhibitor; aninhibitor of the release, or action, of tumor necrosis factor alpha; anantibody therapy, such as monoclonal antibody therapy; an antiviralagent, such as a nucleoside inhibitor (e.g. lamivudine) or an immunesystem modulator (e.g. interferon); an opioid analgesic, such asmorphine; a vanilloid receptor agonist and antagonist; an analgesic,such as a cyclooxygenase-1 and/or cyclooxygenase-2 inhibitor; a localanaesthetic such as lidocaine and derivatives; a stimulant, includingcaffeine; an H2-antagonist (e.g. ranitidine); a proton pump inhibitor(e.g. omeprazole); an antacid (e.g. aluminium or magnesium hydroxide; anantiflatulent (e.g. semethicone); a decongestant (e.g. phenylephrine,phenylpropanolamine, pseudoephedrine, oxymetazoline, epinephrine,naphazoline, xylometazoline, propylhexedrine, or levo-desoxyephedrine,naphazoline, xylometazoline, propylhexedrine, or levo-desoxyephedrine);antitussive (e.g. codeine, hydrocodone, carmiphen, carbetapentane, ordextramethorphan); a diuretic; or a sedating or non-sedatingantihistamine; an antipsychotic agent, including typical and atypicalantipsychotics (e.g. haloperidol, risperidone, clozapine); ananti-depressant, such as a selective serotonin re-uptake inhibitors,serotonin and noradrenaline re-uptake inhibitors, MAO inhibitors andtryciclics antidepressant drugs; a mood stabilizer (e.g. lithium,lamotrigine, valproate); an anxiolytic agent (e.g. benzodiazepines,buspirone, beta-adrenergic receptors antagonists); morphine or morphinederivatives; other calcium or sodium channel blockers.

It is to be understood that the present invention covers the use of acompound of formula (I) or a pharmaceutically acceptable salt thereof incombination with one or more therapeutic agents.

The compounds of the present invention are useful in human andveterinary medicaments. It is to be understood that as used herein theterms “treatment” or “treating” whenever not specifically definedotherwise, include prevention, alleviation and cure of pathologicalafflictions, in particular, they include both the treatment ofestablished symptoms and prophylactic treatment. Compounds of thepresent disclosure may preferably be used therapeutically andpreventively as active ingredients in a pharmaceutical composition.Therefore, a further object of the present invention are pharmaceuticalcompositions containing a therapeutically effective amount of a compoundof the invention or a salt thereof in admixture with a pharmaceuticallyacceptable carrier.

Accordingly, the expression “therapeutically effective” in the contextof an “amount”, a “dose” or “dosage” of the compounds of this inventionis intended as an “amount”, a “dose” or “dosage” of any said compoundssufficient for use in both the treatment of the established symptoms andthe prophylactic treatment of the above said pathological afflictions.

The pharmaceutical compositions object of the present invention may beadministered in a variety of immediate and modified release dosageforms, e.g. orally, in the form of tablets, troches, capsules, sugar orfilm coated tablets, liquid solutions, emulsions or suspensions;rectally, in the form of suppositories; parenterally, e.g. byintramuscular and/or depot formulations; intravenous injection orinfusion; locally and transdermally in form of patch and gel and cream.

Suitable pharmaceutically acceptable, therapeutically inert organicand/or inorganic carrier materials useful in the preparation of suchcomposition include, for example, water, gelatin, gum arabic, lactose,starch, cellulose, magnesium stearate, talc, vegetable oils,cyclodextrins, polyalkyleneglycols and the like.

The composition comprising the phenylethylamino derivatives of formula(I) as above defined can be sterilized and may contain further wellknown components, such as, for example, preservatives, stabilizers,wetting or emulsifying agents, e.g. paraffin oil, mannide monooleate,salts to adjust osmotic pressure, buffers and the like.

For example, the solid oral forms may contain, together with the activeingredient, diluents, e.g. lactose, dextrose, saccharose, cellulose,corn starch or potato starch; lubricants, e.g. silica, talc, stearicacid, magnesium or calcium stearate, and/or polyethylene glycols;binding agents, e.g. starches, arabic gums, gelatin, methylcellulose,carboxymethylcellulose or polyvinyl pyrrolidone; desegregating agents,e.g. a starch, alginic acid, alginates or sodium starch glycolate;effervescing mixtures; dyestuffs; sweeteners; wetting agents such aslecithin, polysorbates, laurylsulphates; and, in general, non-toxic andpharmacologically inactive substances used in pharmaceuticalformulations. Said pharmaceutical preparations may be manufactured inknown manner, for example, by means of mixing, granulating, tabletting,sugar-coating, or film-coating processes.

The preparation of the pharmaceutical compositions object of theinvention can be carried out according to common techniques.

The oral formulations comprise sustained release formulations that canbe prepared in conventional manner, for instance by applying an entericcoating to tablets and granules.

The liquid dispersion for oral administration may be e.g. syrups,emulsions and suspensions. The syrups may contain as carrier, forexample, saccharose or saccharose with glycerine and/or mannitol and/orsorbitol.

Suspensions and emulsions may contain as a carrier, for example, anatural gum, agar, sodium alginate, pectin, methylcellulose,carboxymethyl-cellulose, or polyvinyl alcohol. The suspensions orsolutions for intramuscular injections may contain, together with theactive compound, a pharmaceutically acceptable carrier, e.g. sterilewater, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and, ifdesired, a suitable amount of lidocaine hydrochloride. The solutions forintravenous injections or infusion may contain as carrier, for example,sterile water or preferably they may be in the form of sterile, aqueous,isotonic saline solutions.

The suppositories may contain, together with the active ingredient, apharmaceutically acceptable carrier, e.g. cocoa butter, polyethyleneglycol, a polyoxyethylene sorbitan fatty acid ester surfactants orlecithin.

The pharmaceutical compositions comprising the phenylethylaminoderivatives of formula (I) as above defined will contain, per dosageunit, e.g., capsule, tablet, powder injection, teaspoonful, suppositoryand the like from about 0.1 to about 500 mg of one or more activeingredients most preferably from 1 to 10 mg.

Optimal therapeutically effective doses to be administered may bereadily determined by those skilled in the art and will vary, basically,with the strength of the preparation, with the mode of administrationand with the advancement of the condition or disorder treated. Inaddition, factors associated with the particular subject being treated,including subject age, weight, diet and time of administration, willresult in the need to adjust the dose to an appropriate therapeuticallyeffective level.

It is to be understood that while the invention is described inconjunction of the preferred embodiments thereof, those skilled in theart are aware that other embodiment could be made without departing fromthe spirit of the invention.

EXPERIMENTAL PART

The ¹H-NMR spectra have been determined in solution of CDCl₃ or DMSO-d₆with a Varian Gemini 200 MHz spectrometer. The chemical shifts aredefined as δ with CDCl₃ or DMSO-d₆ and D₂O as inner standard.

The HPLC/MS analyses are determined with a Gilson instrument byutilizing a X-Terra RP18 column (5 μm, 4.6×50 mm) coupled to a UVdetector (220 nm) and a Finnigan Aqa mass spectrometer (electron spray,positive ionization mode). Conditions utilized for the analyses: flow:1.2 ml/min; column temperature: 50° C.; A/B elution gradient (eluent A:0.1% formic acid in water; eluent B: 0.1% formic acid in acetonitrile):5-95% of B from 0 to 8.0 minutes, 95% of B from 8.0 to 9.5 minutes.

For better illustrating the invention the following examples are nowgiven.

Example 1 2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dimethylacetamidehydrochloride

The compound was synthesized according to Scheme I

Step 1: 2-(3-Hydroxyphenyl)-(tert-butoxycarbonyl)ethylamine

Method A

To a suspension of 2-(3-benzyloxyphenyl)-ethylamine hydrochloride (12.6g, 47.7 mmol) in H₂O (120 ml) and 1M NaOH (95 ml), a solution of boc₂O(15.6 g, 71.5 mmol) in THF (120 ml) was added dropwise and the mixturewas stirred at room temperature. After 16 h the organic solvent wasremoved under reduced pressure and the mixture was extracted with CH₂Cl₂(2×100 ml). The collected organic phases were dried over Na₂SO₄, thesolution was filtered and the solvent evaporated under reduced pressureto give a crude oil that was used without further purification.

¹H NMR (300 MHz, CDCl₃): δ 7.47-7.28 (m, 5H), 7.22 (m, 1H), 6.87-6.77(m, 3H), 5.05 (s, 2H), 4.52 (bs, 1H), 3.38 (dt, J=6.5 Hz, J=6.5 Hz, 2H),2.77 (t, J=7.1 Hz, 2H), 1.44 (s, 9H).

ESI⁺MS: calcd for C₂₀H₂₅NO₃: 327.43. found: 328.1 (MH⁺).

The 2-(3-Benzyloxyphenyl)-(tert-butoxycarbonyl)ethylamine obtained inStep 1 and 10% Pd/C (1.3 g) in MeOH (240 ml), was hydrogenated in a Parrapparatus for 16 h at 35 psi. The catalyst was removed by filtration onCelite pad and the solvent was evaporated under reduced pressure. Thecrude oil was used without further purification.

¹H NMR (300 MHz, CDCl₃): δ 7.19 (dd, J=7.8 Hz, J=7.8 Hz, 1H), 6.82-6.66(m, 3H), 4.56 (bs, 1H), 3.39 (dt, J=7.0 Hz, J=6.3 Hz, 2H), 2.76 (t,J=7.0 Hz, 2H), 1.46 (s, 9H).

ESI⁺MS: calcd for C₁₃H₁₉NO₃: 237.30. found: 238.2 (MH⁺).

Method B

A 33% solution of HBr in acetic acid (150 ml) was cooled to 0° C. and3-methoxy phenethylamine (10.0 g, 66.0 mmol) was added portionwise. Themixture was heated to 80° C. and stirred for 16 h. The solvent wasevaporated under reduced pressure and the residue was dissolved in water(160 ml). 4 M NaOH (15 ml) was added followed by 2 M of NaOH (130 ml). Asolution of boc₂O (15.8 g, 72.6 mmol) in THF (160 ml) was added dropwiseand the mixture was stirred at room temperature for 16 h. The upperorganic layer of the resulting mixture was separated and the aqueouslayer was extracted with CH₂Cl₂ (3×100 ml). The combined organicsolutions were dried over Na₂SO₄, filtered and the solvent wasevaporated under reduced pressure. The crude title compound (16.8 g) wasobtained as a brown gum that was used in the following steps withoutfurther purification.

ESI⁺MS: calcd for C₁₃H₁₉NO₃: 237.3. found: 182.1 (MH⁺-t-butyl, majorfragment).

Step 2: 2-(3-Butoxyphenyl)-(tert-butoxycarbonyl)ethylamine

To a solution in acetone (240 ml), of2-(3-hydroxyphenyl)-(tert-butoxycarbonyl)ethylamine obtained in Step 1,K₂CO₃ (19.8 g) and 1-bromobutane (15 ml) were added. The suspension wasrefluxed for 3 days and the solvent was evaporated under reducedpressure. The residue was dissolved in H₂O (200 ml) and extracted withCH₂Cl₂ (2×200 ml). The solvent was eliminated under reduced pressure andthe residue was purified by flash chromatography (petroleum ether/EtOAc85:15) affording 1 (11.3 g, 81% over 3 steps) of the title compound ascolorless oil.

¹H NMR (300 MHz, CDCl₃): δ 7.22 (dd, J=7.6 Hz, J=7.6 Hz, 1H), 6.81-6.72(m, 3H), 4.55 (bs, 1H), 3.97 (t, J=6.3 Hz, 2H), 3.39 (dt, J=6.5 Hz,J=6.5 Hz, 2H), 2.78 (t, J=7.1 Hz, 2H), 1.78 (m, 2H), 1.51 (m, 2H), 1.45(s, 9H), 0.99 (t, J=7.3 Hz, 3H).

ESI⁺MS: calcd for C₁₇H₂₇NO₃: 293.41. found: 294.1 (MH⁺).

Step 3:2-[2-(3-Butoxyphenyl)-(tert-butoxycarbonyl)ethylamino]-N,N-dimethylacetamide

To a suspension of NaH (60%, 2.0 g, 51 mmol) in dry DMF (125 ml) cooledat 0° C., a solution of2-(3-butoxyphenyl)-(tert-butoxycarbonyl)ethylamine (7.5 g, 25.5 mmol) indry DMF (125 ml) was added dropwise. After 1 h at room temperature,2-chloro-N,N-dimethylacetamide (5.2 ml, 51 mmol) was added and themixture was stirred for 16 h at room temperature. H₂O (10 ml) was addedand the solvent was evaporated under reduced pressure. The residue wasdissolved in H₂O (150 ml) and extracted with CH₂Cl₂ (2×150 ml). Thecollected organic phases were dried over Na₂SO₄, filtered andevaporated. The crude was purified by flash chromatography (petroleumether/EtOAc 4:6) affording the title compound (7.2 g, 75%) as lightyellow oil.

¹H NMR (300 MHz, DMSO-d6): δ 7.1 (m, 1H), 6.79-6.71 (m, 3H), 3.97 (t,J=6.0 Hz, 2H), 3.96 (s, 2H), 3.40 (dd, J=8.7 Hz, J=7.2 Hz, 2H), 2.88 (s,6H), 2.76 (dd, J=7.9 Hz, J=6.4 Hz, 2H), 1.76 (m, 2H), 1.46 (m, 2H), 1.37(s, 9H), 0.95 (t, J=7.3 Hz, 3H).

ESI⁺MS: calcd for C₂₁H₃₄N₂O₄: 378.52. found: 379.0 (MH⁺).

Step 4: 2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dimethylacetamidehydrochloride

A solution of2-[2-(3-butoxyphenyl)-(tert-butoxycarbonyl)ethylamino]-N,N-dimethylacetamide(9.6 g, 25.3 mmol) in HCl/Et₂O (127 ml) was stirred at room temperaturefor 16 h. The solvent was evaporated under reduced pressure, the residuewas ground with a mixture of Et₂O/iPr₂O 50/50, filtered and washed withEt₂O/iPr₂O to obtain the title compound as white solid (1.71 g, yield95%).

¹H NMR (300 MHz, CDCl₃): δ 9.63 (br. s., 1H), 7.23 (dd, 1H), 6.83-6.91(m, 2H), 6.80 (ddd, 1H), 3.96 (s, 2H), 3.96 (t, 2H), 3.32-3.44 (m, 2H),3.22-3.32 (m, 2H), 2.97 (s, 6H), 1.70-1.83 (m, 2H), 1.41-1.58 (m, 2H),0.99 (t, 3H).

ESI⁺MS: calcd for C₁₆H₂₆N₂O₂: 278.40. found: 279.3 (MH⁺).

Analogously, starting from the appropriate intermediates, the followingcompounds were prepared:

2-[2-[3-(4,4,4-Trifluorobutoxy)phenyl]-ethylamino]-N,N-dimethylacetamidehydrochloride

¹H NMR (300 MHz, DMSO-d6): δ 9.00 (br. s., 2H), 7.17-7.32 (m, 1H),6.78-6.94 (m, 3H), 4.04 (br. s., 2H), 3.91-4.20 (m, 2H), 3.08-3.22 (m,2H), 2.93-3.00 (m, 2H), 2.94 (s, 3H), 2.90 (s, 3H), 2.30-2.48 (m, 2H),1.78-2.05 (m, 2H).

ESI⁺MS: calcd for C₁₆H₂₃F₃N₂O₂ (free base): 332.27. found: 333.25 (MH⁺).

2-[2-(3-Pentyloxyphenyl)-ethylamino]-N,N-dimethylacetamide hydrochloride

¹H NMR (300 MHz, DMSO-d6): δ 9.03 (br s, 2H), 7.14-7.29 (m, 1H),6.70-6.88 (m, 3H), 4.03 (s, 2H), 3.95 (t, 2H), 3.06-3.21 (m, 2H), 2.94(s, 3H), 2.90 (s, 3H), 2.81-3.02 (m, 2H), 1.62-1.80 (m, 2H), 1.23-1.48(m, 4H), 0.90 (t, 3H).

ESI⁺MS: calcd for C₁₇H₂₈N₂O₂ (free base): 292.42. found: 293.25 (MH⁺).

2-[2-(3-Hexyloxyphenyl)-ethylamino]-N,N-dimethylacetamide hydrochloride

¹H NMR (300 MHz, DMSO-d6): δ 9.01 (br s, 2H), 7.23 (dd, 1H), 6.65-6.93(m, 3H), 4.03 (s, 2H), 3.95 (t, 2H), 3.05-3.24 (m, 2H), 2.94 (s, 3H),2.91-3.01 (m, 2H), 2.90 (s, 3H), 1.57-1.84 (m, 2H), 1.35-1.51 (m, 2H),1.22-1.36 (m, 4H), 0.70-1.01 (m, 3H).

ESI⁺MS: calcd for C₁₈H₃₀N₂O₂ (free base): 306.45. found: 307.32 (MH⁺).

2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dipropylacetamide hydrochloride

¹H NMR (300 MHz, DMSO-d6): δ 8.88 (br. s., 2H), 7.15-7.30 (m, 1H),6.68-6.88 (m, 3H), 4.02 (s, 2H), 3.96 (t, 2H), 3.23-3.28 (m, 2H),3.09-3.22 (m, 4H), 2.87-2.98 (m, 2H), 1.62-1.75 (m, 2H), 1.35-1.62 (m,6H), 0.94 (t, 3H), 0.85 (dt, 6H).

ESI⁺MS: calcd for C₂₀H₃₄N₂O₂ (free base): 334.50. found: 335.34 (MH⁺).

2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dibutylacetamide hydrochloride

¹H NMR (300 MHz, DMSO-d6+TFA): δ 8.85 (br. s., 2H), 7.19-7.28 (m, 1H),6.62-6.88 (m, 3H), 4.01 (t, 2H), 3.96 (t, 2H), 3.30 (t, 2H), 3.06-3.24(m, 4H), 2.85-3.00 (m, 2H), 1.61-1.82 (m, 2H), 1.40-1.55 (m, 6H),1.20-1.38 (m, 4H), 0.94 (t, 6H), 0.89 (t, 3H).

ESI⁺MS: calcd for C₂₀H₃₈N₂O₂ (free base): 362.55. found: 363.35 (MH⁺).

2-[2-(3-Pentyloxyphenyl)-ethylamino]-N,N-dipropylacetamide hydrochloride

¹H NMR (300 MHz, DMSO-d6+Na₂CO₃): δ 7.50 (br. s., 1H) 7.07-7.24 (m, 1H)6.62-6.83 (m, 3H) 3.93 (t, 2H) 3.06-3.22 (m, 5H) 2.58-2.81 (m, 5H)1.62-1.78 (m, 2H) 1.28-1.56 (m, 8H) 0.68-0.99 (m, 9H).

ESI⁺MS: calcd for C₂₁H₃₆N₂O₂ (free base): 348.53. found: 349.28 (MH⁺).

2-[2-(3-Butoxyphenyl)-ethylamino]-acetamide hydrochloride

¹H NMR (300 MHz, DMSO-d6): δ 8.96 (br s, 2H), 7.87 (br s, 1H), 7.54 (brs, 1H), 7.23 (dd, 1H), 6.58-6.83 (m, 3H), 3.96 (t, 2H), 3.70 (s, 2H),3.04-3.18 (m, 2H), 2.82-3.01 (m, 2H), 1.57-1.80 (m, 2H), 1.32-1.54 (m,2H), 0.81-1.04 (m, 3H).

ESI⁺MS: calcd for C₁₄H₂₂N₂O₂: 250.34. found: 251.1 (MH⁺).

2-[2-(3-Butoxyphenyl)-ethylamino]-N-methylacetamide hydrochloride

¹H NMR (300 MHz, DMSO-d6): δ 8.99 (br s, 2H), 8.39 (q, 1H), 7.08-7.37(m, 1H), 6.65-6.95 (m, 3H), 3.96 (t, 2H), 3.70 (s, 2H), 3.04-3.25 (m,2H), 2.79-3.04 (m, 2H), 2.67 (d, 3H), 1.57-1.82 (m, 2H), 1.44 (sxt, 2H),0.94 (t, 3H).

ESI⁺MS: calcd for C₁₅H₂₄N₂O₂: 264.37. found: 265.2 (MH⁺).

2-[2-(3-Isopropoxyphenyl)-ethylamino]-N,N-dimethylacetamidehydrochloride

¹H NMR (300 MHz, DMSO-d6): δ 9.04 (br s, 2H), 7.12-7.32 (m, 1H),6.70-6.81 (m, 3H), 4.60 (spt, 1H), 4.03 (s, 2H), 3.04-3.21 (m, 2H), 2.94(s, 3H), 2.91-3.01 (m, 2H), 2.90 (s, 3H), 1.26 (d, 6H).

ESI⁺MS: calcd for C₁₅H₂₄N₂O₂: 264.37. found: 265.2 (MH⁺).

2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-diethylacetamide hydrochloride

¹H NMR (300 MHz, DMSO-d6): δ 8.89-8.98 (br s, 2H), 7.24 (dd, 1H),6.72-6.88 (m, 3H), 4.03 (s, 2H), 3.96 (t, 2H), 3.34 (q, 2H), 3.26 (q,2H), 3.08-3.21 (m, 2H), 2.86-3.03 (m, 2H), 1.59-1.78 (m, 2H), 1.33-1.54(m, 2H), 1.13 (t, 3H), 1.07 (t, 3H), 0.94 (t, 3H).

ESI⁺MS: calcd for C₁₈H₃₀N₂O₂: 306.45. found: 307.2 (MH⁺).

2-[2-(3-Butoxyphenyl)-ethylamino]-1-pyrrolidin-1-yl-ethanone

¹H NMR (300 MHz, DMSO-d6): δ 8.94 (s, 2H), 7.14-7.33 (m, 1H), 6.64-6.93(m, 3H), 4.00 (t, 2H), 3.88 (s, 2H), 3.33-3.45 (m, 4H), 3.19-3.29 (m,2H), 2.96-3.05 (m, 2H), 1.78-2.00 (m, 4H), 1.65-1.78 (m, 2H), 1.37-1.56(m, 2H), 0.96 (t, 3H).

ESI⁺MS: calcd for C₁₈H₂₈N₂O₂: 304.44. found: 305.2 (MH⁺).

2-[2-(3-Butoxy-4-fluorophenyl)-ethylamino]-N,N-dimethylacetamidehydrochloride

¹H NMR (300 MHz, DMSO-d6): δ 8.91 (br s, 2H) 7.15 (dd, 1H) 7.05 (dd, 1H)6.79 (ddd, 1H) 3.97-4.12 (m, 4H) 3.09-3.21 (m, 2H) 2.94 (s, 3H)2.92-2.99 (m, 2H) 2.90 (s, 3H) 1.65-1.82 (m, 2H) 1.36-1.54 (m, 2H)0.87-1.01 (m, 3H).

ESI⁺MS: calcd for C₁₆H₂₅N₂O₂ (free base): 296.38. found: 297.22 (MH⁺).

2-[2-(3-Butoxy-4-methoxyphenyl)-ethylamino]-N,N-dimethylacetamidehydrochloride

¹H NMR (300 MHz, DMSO-d6): δ 8.97 (br s, 2H), 6.90 (d, 1H), 6.84 (d,1H), 6.74 (dd, 1H), 4.02 (br s, 2H), 3.94 (t, 2H), 3.73 (s, 3H),3.04-3.22 (m, 2H), 2.94 (s, 3H), 2.90 (s, 3H), 2.84-2.92 (m, 2H),1.57-1.84 (m, 2H), 1.44 (sxt, 2H), 0.94 (t, 3H).

ESI⁺MS: calcd for C₁₇H₂₈N₂O₃ (free base): 308.42. found: 309.21 (MH⁺).

Example 2 2-[2-(3-Butoxyphenyl)-ethylamino]-2,N,N-trimethylpropanamidehydrochloride

Step 1: 2-[2-(3-Butoxyphenyl)-ethylamino]-2-methylpropionic acid ethylester To a solution of 2-(3-butoxyphenyl)-ethylamine hydrochloride (0.27g, 1.42 mmol; obtained from the compound of Step 2 of Example 1,according to the standard procedure described in Step 4 of Example 1) inacetonitrile (8 ml), 2-bromo-2-methylpropionic acid ethyl ester (0.27ml, 1.85 mmol) and triethylamine (0.52 ml, 3.70 mmol) were added. Thesolution was heated at 100° C. under microwave irradiation for 3 h. Themixture was cooled to room temperature and partitioned between water andCH₂Cl₂. The organic layer was separated, washed with brine, dried overNa₂SO₄, filtered and the solvent was evaporated under reduced pressure.The residue was purified by flash chromatography (silica, CH₂Cl₂:MeOHfrom 100:0 to 95:5) affording the title compound (0.17 g, 39% yield) ascolourless oil.

ESI⁺MS: calcd for C₁₈H₂₉NO₃: 307.44. found: 308.2 (MH⁺).

Step 2: 2-[2-(3-Butoxyphenyl)-ethylamino]-2,N,N-trimethylpropanamidehydrochloride

To a solution of dimethylamine, 2 M in THF (0.6 ml, 1.1 mmol) in toluene(3 ml) trimethylaluminium, 2 M in hexane (1.4 ml, 2.77 mmol) was addedand the mixture was stirred at room temperature for 15 minutes.2-[2-(3-butoxyphenyl)-ethylamino]-2-methylpropionic acid ethyl ester(0.17 g, 0.55 mmol) in dry toluene (8 ml) was added and the solution washeated to 90° C. and stirred for 24 h. The mixture was cooled to roomtemperature and the solved removed under reduced pressure. The residuewas partitioned between water and diethyl ether. The organic layer wasseparated, washed with brine, dried over Na₂SO₄, filtered and thesolvent was evaporated under reduced pressure. The residue was purifiedby flash chromatography (first purification: silica, CH₂Cl₂:MeOH from100:0 to 97:3; second purification: silica, EtOAc) affording the titlecompound that was dissolved in HCl/Et₂O and stirred for 20 minutes. Theresulting hydrochloride salt was filtered, washed with iPr₂O and driedat 40° C. under vacuum. The pure title compound (18.5 mg, 20% yield) wasobtained as a white solid.

¹H NMR (300 MHz, CDCl₃): δ 9.39 (bs, 2H), 7.24 (t, 1H), 6.71-6.97 (m,3H), 3.97 (t, 2H), 3.13-3.36 (m, 4H), 3.09 (s, 6H), 1.90 (s, 6H),1.69-1.86 (m, 2H), 1.51 (sxt, 2H), 0.99 (t, 3H).

ESI⁺MS: calcd for C₁₈H₃₀N₂O₂: 306.45. found: 307.32 (MH⁺).

Example 3 2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dimethylpropanamidehydrochloride Step 1:2-[2-(3-Butoxyphenyl)-(tert-butoxycarbonyl)ethylamino]-N,N-dimethylpropanamide

A solution of2-[2-(3-butoxyphenyl)-(tert-butoxycarbonyl)ethylamino]-N,N-dimethylacetamide(0.410 g, 1.1 mmol; prepared according to Step 3 of Example 1) in dryTHF (5 ml) was cooled to −78° C. and LiHMDS (lithiumhexamethyldisilazide), 1 M in THF, (1.43 ml, 1.4 mmol) was addeddropwise. The mixture was stirred 30 min then a solution of methyliodide (0.187 g, 1.3 mmol) in dry THF (1 ml) was added dropwise. Themixture was stirred for 2 h allowing the cooling bath to expire. Thesolvent was evaporated under reduced pressure and the residue wasdissolved in EtOAc (15 ml) and washed with water (2×15 ml). The organicphase was dried over Na₂SO₄, filtered and the solvent was evaporatedunder reduced pressure. The residue was purified by flash chromatography(silica, petroleum ether:EtOAc from 8:2 to 6:4) affording the titlecompound (0.22 g, 52% yield) as a colourless oil.

ESI⁺MS: calcd for C₂₂H₃₆N₂O₄: 392.54. found: 393.3 (MH⁺).

Step 2: 2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dimethylpropanamidehydrochloride

The title compound was prepared from2-[2-(3-butoxyphenyl)-(tert-butoxycarbonyl)ethylamino]-N,N-dimethyl-propanamideaccording to the standard procedure described in Step 4 of Example 1.White solid (47% yield).

¹H NMR (300 MHz, DMSO-d6): δ 9.16 (br s, 1H), 8.82 (br s, 1H), 7.14-7.31(m, 1H), 6.64-6.93 (m, 3H), 4.39 (q, 1H), 3.96 (t, 2H), 3.08-3.22 (m,1H), 3.00 (s, 3H), 2.96-3.06 (m, 1H), 2.87-2.96 (m, 2H), 2.90 (s, 3H),1.59-1.82 (m, 2H), 1.37-1.53 (m, 2H), 1.38 (d, 3H), 0.94 (t, 3H).

ESI⁺MS: calcd for C₁₇H₂₈N₂O₂ (free base): 292.42. found: 293.25 (MH⁺).

Example 42-[2-(3-Butoxyphenyl)-ethylamino]-3-hydroxy-N,N-dimethylpropanamidehydrochloride Step 1:2-[2-(3-Butoxyphenyl)-(tert-butoxycarbonyl)ethylamino]-3-acetoxy-N,N-dimethyl-propanamide

The title compound was prepared from[2-[2-(3-butoxyphenyl)-(tert-butoxycarbonyl)ethylamino]-N,N-dimethylacetamideand acetic acid bromomethyl ester according to Step 1 of Example 3.Colourless oil (38% yield).

ESI⁺MS: calcd for C₂₄H₃₈N₂O₆ (free base): 450.58. found: 451.2 (MH⁺).

Step 2:2-[2-(3-Butoxyphenyl)-(tert-butoxycarbonyl)ethylamino]-3-hydroxy-N,N-dimethyl-propanamide

2-[2-(3-Butoxyphenyl)-(tert-butoxycarbonyl)ethylamino]-3-acetoxy-N,N-dimethylpropanamide(0.19 g, 0.42 mmol) was dissolved in 3% NH₄OH/MeOH (15 ml) and stirredat room temperature for 5 h. The solvent was evaporated under reducedpressure and the residue was purified by flash chromatography (silica,petroleum ether:EtOAc from 7:3 to 3:7) affording the title compound(0.13 g, 66% yield) as a colourless oil.

ESI⁺MS: calcd for C₂₂H₃₆N₂O₅: 408.54. found: 409.2 (MH⁺).

Step 3:2-[2-(3-Butoxyphenyl)-ethylamino]-3-hydroxy-N,N-dimethylpropanamidehydrochloride

The title compound was prepared from2-[2-(3-butoxyphenyl)-(tert-butoxycarbonyl)ethylamino]-3-acetoxy-N,N-dimethylpropanamideaccording to the standard procedure described in Step 4 of Example 1.Obtained as white solid (76% yield).

¹H NMR (300 MHz, DMSO-d6): δ 8.46-9.44 (m, 2H) 7.10-7.33 (m, 1H)6.65-6.92 (m, 3H) 5.54 (t, 1H) 4.44 (t, 1H) 3.95 (t, 2H) 3.65-3.88 (m,2H) 3.11-3.22 (m, 1H) 3.03-3.09 (m, 1H) 3.02 (s, 3H) 2.91-2.99 (m, 2H)2.90 (s, 3H) 1.61-1.79 (m, 2H) 1.35-1.53 (m, 2H) 0.86-1.00 (m, 3H).

ESI⁺MS: calcd for C₁₇H₂₈N₂O₃ (free base): 308.42. found: 309.21 (MH⁺).

Example 52-[2-(3-Butoxy-4-methylphenyl)-ethylamino]-N,N-dimethylacetamidehydrochloride Step 1: (3-Hydroxy-4-methylphenyl)-acetonitrile

To a solution of 3-methoxy-4-methylphenylacetonitrile (2.0 g, 12.4 mmol)in dry CH₂Cl₂ (50 ml) cooled to −78° C., BBr₃ 1 M in CH₂Cl₂ (27 ml, 27mmol) was added dropwise. The mixture was stirred overnight allowing thecooling bath to expire. The mixture was slowly poured in ice/water understirring. When the ice melted, the aqueous phase was extracted withEtOAc and the organic phase was dried over Na₂SO₄, filtered and thesolvent was evaporated under reduced pressure. The crude title compound(1.7 g, 93% yield) was used in the next step without furtherpurification.

Step 2: (3-Butoxy-4-methylphenyl)-acetonitrile

To a solution of (3-hydroxy-4-methylphenyl)-acetonitrile (1.7 g, 11.5mmol) in acetone (100 ml), K₂CO₃ (7.9 g, 57.5 mmol) and 1-bromo butane(6.1 ml, 57.5 mmol) were added. The suspension was refluxed for 24 h andthe solvent was evaporated under reduced pressure. The residue wasdissolved in CH₂Cl₂ and washed with water and brine. The organic layerwas dried over Na₂SO₄, filtered and the solvent evaporated under reducedpressure. The crude was purified by flash chromatography (petroleumether:EtOAc 9.5:0.5) affording the title compound (1.43 g, 61% yield) ascolourless oil.

ESI⁺MS: calcd for C₁₃H₁₇NO: 203.29. found: 204.1 (MH⁺).

Step 3: N-[2-(3-Butoxy-4-methylphenyl)-ethyl]-carbamic acid tert-butylester

To a solution of (3-butoxy-4-methylphenyl)acetonitrile (0.94 g, 5.0mmol) in methanol (38 ml), nickel chloride hexahydrate (0.12 g, 0.5mmol) and boc₂O (2.18 g, 10.0 mmol) were added. The solution was cooledto 0° C. and sodium borohydride (1.32 g, 35.0 mmol) was addedportionwise over 30 minutes. The mixture was stirred overnight allowingthe cooling bath to expire. The reaction was quenched by addition ofdiethylenetriamine (0.54 ml, 5 mmol) and stirred for 30 minutes. Thesolvent was removed under reduced pressure and the residue re-dissolvedin EtOAc, washed with water, dried over Na₂SO₄, filtered and the solventwas evaporated under reduced pressure. The crude was purified by flashchromatography (silica, petroleum ether:EtOAc 90:10) to give the titlecompound. Colourless oil (80% yield).

ESI⁺MS: calcd for C₁₈H₂₉NO₃: 307.44. found: 308.1 (MH⁺).

Step 4: 2-[2-(3-Butoxy-4-methylphenyl)-ethylamino]-N,N-dimethylacetamidehydrochloride

The title compound was prepared from[2-(3-butoxy-4-methylphenyl)-ethyl]-carbamic acid tert-butyl esteraccording to the standard procedures described in Steps 3 and 4 ofExample 1. Obtained as white solid.

¹H NMR (300 MHz, DMSO-d6): δ 8.93 (br s, 2H), 7.08 (dd, 1H), 6.79 (d,1H), 6.69 (dd, 1H), 4.03 (s, 2H), 3.97 (t, 2H), 3.08-3.19 (m, 2H), 2.93(s, 3H), 2.91-2.97 (m, 2H), 2.90 (s, 3H), 2.11 (s, 3H), 1.65-1.79 (m,2H), 1.39-1.54 (m, 2H), 0.95 (t, 3H).

ESI⁺MS: calcd for C₁₇H₂₈N₂O₂ (free base): 292.42. found: 293.25 (MH⁺).

Analogously, starting from (2,6-difluoro-3-methoxyphenyl)-acetonitrilethe following compound was prepared:

2-[2-(3-Butoxy-2,6-difluorophenyl)-ethylamino]-N,N-dimethylacetamidehydrochloride

¹H NMR (300 MHz, DMSO-d6): δ 9.16 (br s, 2H), 7.07-7.18 (m, 1H),6.96-7.07 (m, 1H), 4.07 (s, 2H), 4.02 (t, 2H), 3.08 (s, 4H), 2.93 (s,3H), 2.90 (s, 3H), 1.63-1.76 (m, 2H), 1.36-1.51 (m, 2H), 0.93 (t, 3H).

ESI⁺MS: calcd for C₁₆H₂₄F₂N₂O₂ (free base): 314.37. found: 315.20 (MH⁺).

Example 62-[2-(3-Butoxyphenyl)-2-methylpropylamino]-N,N-dimethylacetamidehydrochloride Step 1: 2-(3-Methoxyphenyl)-2-methylpropionitrile

To a solution of (3-methoxyphenyl)-acetonitrile (8.0 g, 0.054 mol) inDMF (25 ml) cooled to 0° C., NaH (1.3 g, 0.054 mol) was added. Thereaction was stirred for 30 min, and MeI (3.3 mL, 0.054 mol) was added.The reaction was stirred 1 h at room temperature. After this period, thereaction mixture was cooled again to 0° C., NaH (1.3 g, 0.054 mol) wasadded followed by MeI (3.3 ml, 0.054 mol) after 30 minutes. The reactionwas stirred at room temperature overnight. DMF was evaporated and thecrude diluted with brine and extracted with Et₂O. The organic phase waswashed with water, dried over Na₂SO₄, the solvent was evaporated underreduced pressure and the residue was purified by flash chromatography(petroleum ether:AcOEt 95:5) to give the title compound (4 g, 42% yield)as a colourless oil.

ESI⁺MS: calcd for C₁₁H₁₃NO: 175.23. found: 176.1 (MH⁺).

Step 2: 2-[2-(3-Butoxyphenyl)-2-methylpropylamino]-N,N-dimethylacetamidehydrochloride

The title compound was prepared from2-(3-methoxyphenyl)-2-methylpropionitrile according to the proceduredescribed in Example 5.

¹H NMR (300 MHz, DMSO-d6): δ 8.44 (br. s., 2H), 7.28 (t, 1H), 6.90-7.04(m, 2H), 6.77-6.90 (m, 1H), 3.99 (t, 2H), 3.88 (s, 2H), 3.16 (s, 2H),2.88 (s, 6H), 1.64-1.78 (m, 2H), 1.42-1.55 (m, 2H), 1.36-1.42 (m, 6H),0.90-1.04 (m, 3H).

ESI⁺MS: calcd for C₁₈H₃₀N₂O₂ (free base): 306.45. found: 307.26 (MH⁺).

Example 7 2-[2-(3-Butylthiophenyl)-ethylamino]-N,N-dimethylacetamidehydrochloride Step 1: (3-Butylthiophenyl)-acetonitrile

To a solution of (3-iodophenyl)-acetonitrile (2.0 g, 8.2 mmol) andacetic acid S-butyl ester (2.4 ml, 24.6 mmol) in n-BuOH (5 ml), CuI(0.156 g, 0.8 mmol), ethylene glycol (0.96 ml, 1.7 mmol) and K₂CO₃ (2.4g, 17.3 mmol) were added and the mixture was heated under microwaveirradiation to 110° C. for 1 h. The reaction mixture was diluted withAcOEt and filtered over a celite pad. The organic phase was washed withwater and brine, dried over Na₂SO₄ and the solvent was evaporated underreduced pressure. The crude was purified by flash chromatography(petroleum ether:AcOEt from 10:0 to 9:1) to give pure title compound asa pale yellow oil (1.0 g, 64% yield), used without further purificationin the next step.

ESI⁺MS: calcd for C₁₂H₁₅NS: 205.32, no mass detectable.

Step 2: 2-[2-(3-Butylthiophenyl)-ethylamino]-N,N-dimethylacetamidehydrochloride

The title compound was prepared from (3-butylthiophenyl)-acetonitrileaccording to the procedure described in Example 5.

¹H NMR (300 MHz, DMSO-d6): δ 9.08 (br. s., 2H), 7.23-7.36 (m, 1H),7.15-7.23 (m, 2H), 6.95-7.11 (m, 1H), 4.03 (s, 2H), 3.08-3.21 (m, 2H),2.92-3.02 (m, 4H), 2.94 (s, 3H), 2.90 (s, 3H), 1.49-1.67 (m, 2H),1.30-1.49 (m, 2H), 0.89 (t, 3H).

ESI⁺MS: calcd for C₁₆H₂₆N₂OS (free base): 294.46. found: 295.20 (MH⁺).

Example 8 2-[2-(3-Butylsulfonylphenyl)-ethylamino]-N,N-dimethylacetamidehydrochloride Step 1:2-[2-(3-Butylsulfonylphenyl)-(tert-butoxycarbonyl)ethylamino]-N,N-dimethylacetamide

To a solution of2-[2-(3-butylthiophenyl)-(tert-butoxycarbonyl)ethylamino]-N,N-dimethylacetamidehydrochloride (0.25 g, 0.62 mmol; prepared from the compound of Step 2of Example 7, by reaction with boc₂O according to the proceduredescribed in the first part of Step 1 of Example 1) in acetonitrile (20ml)/water (10 ml), a mixture of oxone (0.92 g, 1.5 mmol) and NaHCO₃ (0.2g, 2.3 mmol) was added portionwise over 5 minutes. The mixture wasstirred at room temperature for 2 h. The mixture was partitioned betweenwater and CH₂Cl₂, the organic was washed with water and brine, driedover Na₂SO₄ and the solvent was evaporated under reduced pressure. Thecrude was purified by flash chromatography (petroleum ether:AcOEt 3:7)to give pure title compound as a colourless oil (0.20 g, 75% yield).

ESI⁺MS: calcd for C₂₁H₃₄N₂O₅S: 426.58. found: 427.1 (MH⁺).

Step 2: 2-[2-(3-Butylsulfonylphenyl)-ethylamino]-N,N-dimethylacetamidehydrochloride

The title compound was prepared from2-[2-(3-butylsulfonylphenyl)-(tert-butoxycarbonyl)ethylamino]-N,N-dimethylacetamideaccording to the standard procedure described in Step 4 of Example 1.

¹H NMR (300 MHz, DMSO-d6+TFA): δ 8.84-9.10 (m, 2H), 7.74-7.86 (m, 2H),7.57-7.71 (m, 2H), 4.06 (t, 2H), 3.03-3.34 (m, 6H), 2.95 (s, 3H), 2.91(s, 3H), 1.45-1.63 (m, 2H), 1.24-1.42 (m, 2H), 0.84 (s, 3H).

ESI⁺MS: calcd for C₁₆H₂₆N₂OS (free base): 294.46. found: 295.20 (MH⁺).

Example 9 2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dimethylthioacetamidehydrochloride Step 1:2-[2-(3-Butoxyphenyl)-(tert-butoxycarbonyl)ethylamino]-N,N-dimethylthioacetamide

To a solution of2-[2-(3-butoxyphenyl)-(tert-butoxycarbonyl)ethylamino]-N,N-dimethylacetamide(0.38 g, 1.0 mmol) in dry toluene (20 ml), Lawesson's reagent (0.58 g,1.2 mmol) was added one-pot and the mixture was heated to reflux andstirred for 2 h. The solvent was evaporated under reduced pressure andthe residue was purified by flash chromatography (petroleum ether:AcOEtfrom 9:1 to 8:2) to give pure title compound as a colourless oil (0.11g, 28% yield).

ESI⁺MS: calcd for C₂₁H₃₄N₂O₃S: 394.58. found: 395.1 (MH⁺).

Step 2: 2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-dimethylthioacetamidehydrochloride

The title compound was prepared from2-[2-(3-butoxyphenyl)-(tert-butoxycarbonyl)ethylamino]-N,N-dimethylthioacetamideaccording to the standard procedure described in Step 4 of Example 1.Obtained as white solid.

¹H NMR (300 MHz, DMSO-d6): δ 8.88 (s, 2H), 7.16-7.34 (m, 1H), 6.73-6.88(m, 3H), 4.17 (s, 2H), 3.96 (dd, 2H), 3.43 (s, 3H), 3.31 (s, 3H),3.16-3.26 (m, 2H), 2.92-3.04 (m, 2H), 1.62-1.76 (m, 2H), 1.36-1.50 (m,2H), 0.94 (t, 3H).

ESI⁺MS: calcd for C₁₆H₂₆N₂OS (free base): 294.46. found: 295.22 (MH⁺).

Example 102-[2-(3-Butoxyphenyl)]-(N′-methoxy)ethylamino]-N,N-dimethylacetamideStep 1: 2-(3-Butoxyphenyl)-acetaldehyde

To a solution of 2-(3-butoxyphenyl)-ethanol (3.00 g, 15.4 mmol; preparedfrom 2-(3-hydroxyphenyl)-ethanol according to the procedure described inStep 2 of Example 1) in CH₂Cl₂ (100 ml), Dess-Martin periodinane reagent(8.5 g, 20.1 mmol) was added and the reaction was left at roomtemperature overnight. The solution was poured into a NaHCO₃ saturatedsolution containing Na₂S₂O₃ (35 g), and the mixture was stirred for 30min. The organic layer was separated, dried over Na₂SO₄, filtered andevaporated under reduced pressure.

The residue containing the title compound (2.9 g, 99% yield) was used inthe next step without further purification.

Step 2: 2-(3-Butoxyphenyl)-(N-methoxy)ethylamine

To a suspension of 2-(3-butoxypheny)-acetaldehyde (2.00 g, 10.4 mmol)and 0-methoxyamine hydrochloride (1.12 g, 13.4 mmol) in water (13 ml), asolution of Na₂CO₃ (0.66 g, 6.2 mmol) in water (20 ml) was addeddropwise under stirring at 0° C. The reaction was left at roomtemperature overnight and then extracted with diethylether. The organiclayer was dried over Na₂SO₄, filtered and evaporated under reducedpressure.

The residue containing the desired oxime intermediate (2.24 g, 10.2mmol) was dissolved in methanol (60 ml) and acetic acid (8.8 ml, 153.0mmol) was added. The solution was cooled to 0° C. and NaCNBH₃ was addedportionwise. The reaction mixture was stirred at room temperatureovernight, then the solvent was removed under reduced pressure and theresidue was partitioned between 5% NaHCO₃ solution and ethyl acetate.The organic phase was dried over Na₂SO₄, filtered and concentrated todryness under vacuum. The crude residue was purified by columnchromatography, (petroleum ether:ethyl acetate 9:1) to afford 0.88 g(38% yield) of the title compound.

Step 3:2-[2-(3-Butoxyphenyl)]-(N′-methoxy)ethylamino]-N,N-dimethylacetamide

2-(3-Butoxyphenyl)-(N-methoxy)ethylamine (0.5 g, 2.25 mmol), obtained asdescribed in Step 2, was dissolved in acetonitrile (15 ml) andethyldiisopropylamine (1.95 ml, 11.25 mmol) was added followed by2-chloro-N,N-dimethylacetamide (1.15 ml, 11.25 mmol). The solution washeated at 130° C. under microwave irradiation for 6 h. The mixture wascooled to room temperature, the solvent removed under reduced pressureand the residue was partitioned between 5% NaHCO₃ and ethyl acetate. Theorganic phase was dried over Na₂SO₄, filtered and concentrated todryness under vacuum. The crude residue was purified by columnchromatography, (petroleum ether:ethyl acetate 1:1) to afford 0.25 g(36% yield) of the title compound.

¹H NMR (300 MHz, DMSO-d6+TFA): 7.17 (t, 1H), 6.61-6.89 (m, 3H), 3.94 (t,2H), 3.56 (s, 2H), 3.47 (s, 3H), 2.98 (s, 3H), 2.89-2.97 (m, 2H), 2.80(s, 3H), 2.72-2.86 (m, 2H), 1.58-1.77 (m, 2H), 1.34-1.53 (m, 2H), 0.93(t, 3H).

ESI⁺MS: calcd for C₁₇H₂₈N₂O₃ (free base): 308.42. found: 309.18 (MH⁺).

Example 11 TTXs-Sodium Channel Influx Assay

ND7/23 rat dorsal root ganglion-derived cell line endogenously expressesa mixed population of TTXs sodium channels. These cells lack of TTXrsodium channels as shown by the absence of their respective transcripts.

ND7/23 cells were grown in DMEM supplemented with 10% FBS and 1 mMsodium piruvate. The cells were seeded at 50,000 cells/well on 96poly-L-lysine-coated plates and further incubated for 18-24 h beforeuse.

The Membrane Potential Kit Assay (Molecular Devices), based on anegatively charged fluorescent dye able to monitor changes in membranepotential caused by the sodium influx due to the channel opening, wasused for the assay.

Cells were incubated with the dye loading for 30 minutes at 25° C. Then,100 nM of the toxin Anemonia sulcata (used as enhancer of the channelopener response) alone or in the presence of TTX (as reference standard)or test compound were added for further 15 minutes.

The fluorescence (excitation: 530 nm, emission: 565 nm wavelength) wasmeasured before and after (40-45 s) the automated injection of thesodium channel opener veratridine (100 μM) using a Victor plate reader(Perkin Elmer).

The inhibition curves were calculated from 5 concentrations, each intriplicate, and the IC₅₀ determined using linear regression analysis.

The compounds of the present invention inhibit TTXs sodium channels withpharmacologically significant IC₅₀ values.

The results, obtained with some compounds which are representative ofthe entire class of compounds of the invention, compared with thestandards ralfinamide and safinamide, are reported in Table 1.

TABLE 1 Na⁺ influx Compound IC₅₀ μM2-[2-(3-Hexyloxyphenyl)-ethylamino]-N,N- 0.5 dimethylacetamidehydrochloride 2-[2-(3-Butoxyphenyl)-ethylamino]-N,N- 0.5dipropylacetamide hydrochloride2-[2-(3-Pentyloxyphenyl)-ethylamino]-N,N- 0.5 dimethylacetamidehydrochloride 2-[2-(3-Butoxyphenyl)-ethylamino]-N,N- 0.6diethylacetamide hydrochloride 2-[2-(3-Butoxyphenyl)-ethylamino]-2,N,N-0.7 trimethylpropanamide hydrochloride2-[2-(3-Butoxy-4-methylphenyl)-ethylamino]- 0.7 N,N-dimethylacetamidehydrochloride 2-[2-(3-Pentyloxyphenyl)-ethylamino]-N,N- 1.1dipropylacetamide hydrochloride 2-[2-(3-Butoxyphenyl)-ethylamino]-N,N-1.1 dimethylpropanamide hydrochloride2-[2-(3-Butoxyphenyl)-ethylamino]-N,N- 1.2 dibutylacetamidehydrochloride 2-[2-(3-Butoxyphenyl)-ethylamino]-N,N- 1.2dimethylthioacetamide hydrochloride2-[2-(3-Butoxyphenyl)-ethylamino]-N,N- 1.5 dimethylacetamidehydrochloride 2-[2-(3-Butoxyphenyl)-ethylamino]-1- 2.1pyrrolidin-1-yl-ethanone hydrochloride2-[2-(3-Butoxy-2,6-difluorophenyl)- 2.6ethylamino]-N,N-dimethylacetamide hydrochloride(S)-(+)-2-[4-(2-Fluorobenzyloxy)- 9.5 benzylamino]-propanamide(ralfinamide)* (S)-(+)-2-[4-(3-Fluorobenzyloxy)- 7.4benzylamino]-propanamide (safinamide)* *as the salt with methanesulfonicacid

Example 12 Calcium Channel Influx Assay

AtT20/D16v-F2 mouse pituitary tumour cell line preferentially expressesL-type calcium channels.

AtT20 cells were grown in DMEM with 10% of FBS, 4 mM glutamine. Thecells were seeded at 200,000 cells/well on 96 poly-L-lysine-coatedplates and further incubated for 18-24 h, before use.

The Ca⁺⁺ Kit Assay (Molecular Devices), which is based on a fluorescentcalcium indicator to detect the calcium influx determined bydepolarizing conditions, was used for the assay.

Cells were incubated with the calcium dye loading for 30 min at 37° C.Then, ω-conotoxin alone (1 μM) or in presence of nifedipine (asreference standard) or test compound were added for further 15 min.

The fluorescence (excitation: 485-emission: 535 nm wavelength) wasmeasured before and after (30-40 sec) the automated injection of 100 mMKCl depolarizing solution using a Victor plate reader (Perkin Elmer).

The inhibition curves were calculated from 5 concentrations, each intriplicate, and the IC₅₀ determined using linear regression analysis.

The results, obtained with some compounds which are representative ofthe entire class of compounds of the invention, compared with thestandards ralfinamide and safinamide, are reported in Table 2.

TABLE 2 Ca⁺⁺ influx Compound IC₅₀ μM2-[2-(3-Butoxyphenyl)-ethylamino]-N,N- 1.1 dipropylacetamidehydrochloride 2-[2-(3-Butoxyphenyl)-ethylamino]-N,N- 1.8dibutylacetamide hydrochloride 2-[2-(3-Hexyloxyphenyl)-ethylamino]-N,N-4.0 dimethylacetamide hydrochloride2-[2-(3-Butoxyphenyl)-ethylamino]-N,N- 6.7 diethylacetamidehydrochloride 2-[2-(3-Pentyloxyphenyl)-ethylamino]-N,N- 6.9dipropylacetamide hydrochloride2-[2-(3-Butylthiophenyl)-ethylamino]-N,N- 7.2 dimethylacetamidehydrochloride 2-[2-(3-Butoxyphenyl)-ethylamino]-N,N- 7.6dimethylthioacetamide hydrochloride2-[2-(3-Pentyloxyphenyl)-ethylamino]-N,N- 8.3 dimethylacetamidehydrochloride 2-{2-[3-(4,4,4-Trifluorobutoxy)phenyl]- 9.3ethylamino}-N,N-dimethylacetamide hydrochloride2-[2-(3-Butoxy-4-methylphenyl)-ethylamino]-N,N- 10.0 dimethylacetamidehydrochloride (S)-(+)-2-[4-(2-Fluorobenzyloxy)-benzylamino]- 26.0propanamide (ralfinamide)*(S)-(+)-2-[4-(3-Fluorobenzyloxy)-benzylamino]- 33.0 propanamide(safinamide)* *as the salt with methanesulfonic acid

Example 13 Patch Clamp Evaluation of Na⁺ Currents Blockade in RatCortical Neurons

Cell Preparation and Culturing

Procedures involving animals and their care were conducted in conformitywith institutional guidelines in compliance with national (D.L. n.116,G.U., suppl.40, Feb. 18, 1992) and international laws and policies (EECCouncil directive 86/609, OJL358.1, Dec. 12, 1987; Guide for the Careand Use of Laboratory Animals, U.S. National Research Council, 1996).Cortical neurons were prepared from embryonic Wistar rats (E17-E19). Afemale rat at date 17-19 of pregnancy was anesthetized and sacrificed.The fetuses (n=4-5) were dissected and placed in ice-cold Hank'ssolution (Hank's solution (Life tech.14170-088)+glucose 30%+Pen-Strep100× (Life Tech. 15140-122) 100 U-100 μg/ml and Hepes-NaOH 5 mM). Theuterus and placenta were removed, the fetuses were decapitated and theheads were placed in ice-cold Hank's solution.

The skin of the head was removed using a pincer, the scalp was openedcutting laterally from the back till the eyes, and the brain was takenout using a curved pincer.

The brain was cut in two halves, the outer connective tissue membranewas removed with a pincer, and, keeping the brain upside down, thecerebellum, the brainstem and the diencephalon was removed using acurved pincer trying to clean as much as possible the inside of thecortex.

Each cortex was cut in smaller parts with a scissors, the pieces weretransferred to a 15 ml centrifuge tube using a 5 ml pipette and washedtwice with Hank's solution.

The solution was removed except 1-2 ml and the tissue was firstdissociated with a 5 ml pipette then with two fire-polished Pasteurpipettes (medium and small opening, respectively). After the mechanicaldissociation, 5 ml of complete DMEM (Dulbecco's modified Eagle medium)(Gibco 41966-029)+FBS (Hyclone) 10%+Glutamine (Life Tech. 25030-024) 2mM+Pen-Strep 100 U-100 μg/ml were added, and cell suspension wascentrifuged for 5 min at 1000 rpm. Supernatant was removed and 5 ml ofcomplete Neurobasal medium was added (NB medium (Lifetech.21103-049)+B27 (Life tech.17504-044) 2%+Glutamine 2 mM+Pen-Strep100 U-100 μg/ml).

Cells were counted and diluted in Neurobasal medium to a concentrationof 400000 cells per poly-D-lysine 5 μg/ml treated Petri dish.

Cortical neurons were used from day 6^(th) till day 11^(th) afterplating, and once a week Neurobasal medium was changed.

Whole Cell Patch Clamp Recordings

Experiments on cortical neurons were carried out using standard wholecell patch clamp methods (Hamill et al., Pfugers Arch., 1981 August391(12), 85-100). Membrane currents were recorded and filtered at 5 kHzwith an Axon Axopatch 200B amplifier and data digitized with an AxonDigidata 1322A (Axon Instruments, CA, USA). Protocol playing and dataacquisition were controlled online with Axon pClamp8 software. Measuringand reference electrodes were AgCl—Ag electrodes. A Sutter InstrumentP-87 Puller (CA, USA) was used for pulling patch clamp pipettes with aresistance of 2-3 MΩ from Harward borosilicate glass tubes. Cells werecontinuously superfused with extracellular solutions, using a solutionchanger Biologic RSC-200.

Voltage Protocols and Data Analyses

To test the effect of compounds on sodium currents in cortical neurons,cells were clamped at −90 mV, then a two step protocol was used todetermine the voltage dependence of the block. Sodium currents wereactivated by a 30 ms step pulse to −10 mV (test pulse) from a 2000 mspreconditioning potential of −110 mV (resting condition) and a potentialof ˜−50 mV (half maximal steady-state condition).

Tonic block of resting and depolarized currents at a given drugconcentration, was calculated as the difference between the peak Na⁺current in the control external bath solution and peak currents with thetest substance divided by control peak.

Drug concentration-inhibition curves were obtained by plotting tonicblocks in the resting and depolarized condition, versus drugconcentrations. Dose-response curves were fitted to the tonic blockdata, according to the logistic equation: y=A2+(A1−A2)/[1+(x/IC₅₀)p]. A1and A2 are fixed values of 0 and 1 corresponding to 0 and 100% currentinhibition, x is the drug concentration, IC₅₀ is the drug concentrationresulting in 50% current inhibition and p is the corresponding slopefactor.

The apparent affinity of drug for the inactivated state (Ki) wascalculated according to the equation 1/Kdep=h/Kr+(1-h)/Ki where Kr isthe affinity of drug for the resting/closed state; Kdep is the IC₅₀ inthe depolarized condition, h and (1-h) are the fractions of channelspresent at the rest and dep potentials, respectively.

Solutions and Drugs

Control bath solution contained (mM): NaCl 60, CholineCl 60, CaCl2 1.3,MgCl2 2, KCl 2, CdCl2 0.4, NiCl2 0.3, TEACl 20, Hepes 10, Glucose 10.

Internal pipette solution consisted of (mM): CsF 65, CsCl 65, NaCl 10,CaCl2 1.3, MgCl2 2, Hepes 10, EGTA 10, MgATP 1.

Compounds were dissolved as stock solutions (20 mM) in DMSO. They werediluted to the final concentrations in the external solution.

Results

The compounds of this invention are able to block Na⁺ currents in ratcortical neurons. The results obtained with2-[2-(3-butoxyphenyl)-ethylamino]-N,N-dimethylacetamide hydrochloride(NW-3509), a representative compound of the chemical class of thisinvention, compared with our standards safinamide and ralfinamide, arereported in Table 3.

TABLE 3 IC₅₀ IC₅₀ de- resting polarized COMPOUND (Kr) (μM) (μM) Ki2-[2-(3-Butoxyphenyl)-ethylamino]-N,N- 25 0.8 0.4 dimethylacetamidehydrochloride (S)-(+)-2-[4-(3-Fluorobenzyloxy)- >100 (180) 7.0 3.6benzylamino]-propanamide (safinamide)*(S)-(+)-2-[4-(2-Fluorobenzyloxy)- >100 (213) 9.0 4.7benzylamino]-propanamide (ralfinamide)* *as the salt withmethanesulfonic acid

Data expressed as IC₅₀ value at μM concentration, as well as theaffinity for the inactivated state (Ki) demonstrate that the compound ofthis invention is a very potent and voltage dependent sodium channelblocker.

Example 14 Patch Clamp Evaluation of Na⁺ Currents Blockade in ND7/23Cell Line

Cell Line Maintenance

ND7/23 (ECACC No 92090903 from SIGMA) is a hybrid cell line derived froma neonatal rat DRG fused with the mouse neuroblastoma N18Tg2 (Wood etal., Proc. Biol. Sci., 1990 September, 241 (1302), 187-194). ND7/23cells exhibit sensory neuron-like properties and expresstetrodotoxin-sensitive (TTX-s) but not tetrodotoxin-resistant (TTX-r)currents (Zhou et al., J. Pharmacol. Exp. Ther., 2003 August, 306(2),498-504; John et al., Neuropharmacology, 2004 March 46(3), 425-438)).The lack of TTX-r currents is consistent with the absence of TTX-rchannel transcripts in these cells.

The molecular identity of the channels responsible for the TTX-sconductance is unknown but it is presumed that TTX-s conductance arisesfrom the activity of a mixed population of sodium channels.

Cells are routinously maintained in DMEM with 10% of FBS, 4 mMglutamine, 1 mM sodium piruvate. The day before the patch clampexperiment cells are detached and seeded at 100,000cells/polylysine-coated 35 mm Petri dish.

Whole Cell Patch Clamp Recordings

Experiments on ND7/23 cells were carried out using standard whole cellpatch clamp methods (Hamill et al., Pfughers Arch., 1981 August 391(12),85-100), as described in previous section.

Voltage Protocols and Data Analyses

To test the effect of compounds on sodium currents in ND7/23 cells,holding membrane potential was set at −90 mV, then a two step protocolwas used to determine the voltage dependence of the block. Sodiumcurrents were activated by a 30 ms step pulse to 0 mV (test pulse) froma 2000 ms preconditioning potential of −110 mV (resting condition) and apotential of ˜−70 mV (half maximal steady-state condition).

Tonic block of resting and depolarized currents at a given drugconcentration was calculated as the difference between the peak Na⁺current in the control external bath solution and peak currents with thetest substance divided by control peak.

Drug concentration-inhibition curves were obtained by plotting tonicblocks in the resting and depolarized condition, versus drugconcentrations. Dose-response curves were fitted to the tonic blockdata, according to the logistic equation: y=A2+(A1-A2)[1+(x/IC₅₀)p]. A1and A2 are fixed values of 0 and 1 corresponding to 0 and 100% currentinhibition, x is the drug concentration, IC₅₀ is the drug concentrationresulting in 50% current inhibition and p is the corresponding slopefactor.

The apparent affinity of drug for the inactivated state (Ki) wascalculated according to the equation 1/Kdep=h/Kr+(1-h)/Ki where Kr isthe affinity of drug for the resting/closed state; Kdep is the IC₅₀ inthe depolarized condition, h and (1-h) are the fractions of channelspresent at the rest. and dep. potentials, respectively.

Solutions and Drugs

Control bath solution contained (mM): NaCl 80, Choline HCl 40, CaCl21.3, MgCl2 2, KCl 2, CdCl2 0.4, NiCl2 0.3, TEACl 20, Hepes 10, Glucose10.

Internal pipette solution consisted of (mM): CsF 65, CsCl 65, NaCl 10,CaCl2 1.3, MgCl2 2, Hepes 10, EGTA 10, MgATP 1.

Compounds were dissolved as stock solutions (20 mM) in DMSO. They werediluted to the final concentrations in the external solution.

Results

The compounds of this invention are able to block Na⁺ currents in ND7/23cells. The results obtained with2-[2-(3-pentyloxyphenyl)-ethylamino]-N,N-dimethylacetamide hydrochlorideNW-3525 and2-[2-(3-butoxy-2-fluorophenyl)-ethylamino]-N,N-diethylacetamide,representative compounds of the chemical class of this invention,compared with our standard ralfinamide, are reported in Table 4.

TABLE 4 IC₅₀ IC₅₀ de- resting polarized COMPOUND (Kr) (μM) (μM) Ki2-[2-(3-Pentyloxyphenyl)-ethylamino]- 2.9 0.7 0.5 N,N-dimethylacetamidehydrochloride 2-[2-(3-Butoxy-2-fluorophenyl)- 13 1.7 1ethylamino]-N,N-dimethylacetamide (S)-(+)-2-[4-(2-Fluorobenzyloxy)-149.0 15.0 8.3 benzylamino]-propanamide (ralfinamide)* *as the salt withmethanesulfonic acid

Data expressed as IC₅₀ value at μM concentration, as well as theaffinity for the inactivated state (Ki) demonstrate that the twocompounds of this invention are is a very potent and voltage dependentsodium channel blockers.

Example 15 In Vitro MAO-B Enzyme Activity Assay

Membrane Preparation (Crude Mitochondrial Fraction)

Male Wistar rats (Harlan, Italy weighing 175-200 g) were sacrificedunder light anaesthesia and brains were rapidly removed and homogenizedin 8 volumes of ice-cold 0.32 M sucrose buffer containing 0.1 M EDTA, pH7.4. The crude homogenate was centrifuged at 2220 rpm for 10 minutes andthe supernatant recovered. The pellet was homogenized and centrifugedagain. The two supernatants were pooled and centrifuged at 9250 rpm for10 minutes at +4° C. The pellet was resuspended in fresh buffer andcentrifuged at 11250 rpm for 10 minutes at +4° C. The resulting pelletwas stored at −80° C.

In Vitro Enzyme Activity Assay

The enzyme activity was assessed with a radioenzymatic assay using thesubstrate ¹⁴C-phenylethylamine (PEA) specific for MAO-B.

The mitochondrial pellet (500 μg protein) was resuspended in 0.1 Mphosphate buffer (pH 7.4). 200 μl of the suspension were added to a 50μl solution of the test compound or buffer, and incubated for 30 min at37° C. (preincubation) then the substrate (50 μl) was added. Theincubation was carried out for 10 minutes at 37° C. (¹⁴C-PEA, 0.5 μM).

The reaction was stopped by adding 0.2 ml of perchloric acid. Aftercentrifugation, the deaminated metabolites were extracted with 3 ml oftoluene and the radioactive organic phase containing the neutral and/oracidic metabolites formed as a result of MAO-B activity was measured byliquid scintillation spectrometry at 90% efficiency.

The MAO-B activity was expressed as nmoles of substrate transformed/mgprotein/min.

Compounds representative of the entire chemical class of this inventiondon't show MAO-B inhibition, at relevant concentrations, as reported inTable 5 as IC₅₀ values (the concentration of the compound able toinhibit by 50% the MAO-B enzyme activity).

As a matter of fact a significative MAO-B inhibition is considered whenthe IC₅₀ values are in the sub-micromolar range such as our standardssafinamide and ralfinamide.

TABLE 5 MAO-B Compound IC₅₀ μM2-[2-(3-Butoxyphenyl)-ethylamino]-N-methylacetamide 1102-[2-(3-Butoxyphenyl)-ethylamino]-N,N- 231 dimethylacetamidehydrochloride 2-[2-(3-Butoxy-2,6-difluorophenyl)-ethylamino]-N,N- >300dimethylacetamide hydrochloride2-[2-(3-Butoxy-4-methoxyphenyl)-ethylamino]-N,N- >300 dimethylacetamide2-[2-(3-Butoxyphenyl)-ethylamino]-N,N- >300 dimethylpropanamidehydrochloride (S)-(+)-2-[4-(3-Fluorobenzyloxy)-benzylamino]- 0.1propanamide (safinamide)* (S)-(+)-2-[4-(2-Fluorobenzyloxy)-benzylamino]-0.2 propanamide (ralfinamide)* *as the salt with methanesulfonic acid

Example 16 Formalin Test

According to a modified protocol from Rosland et al. (Rosland J. H.,Tjolsen A., Maehle B., Hole K. Pain (1990) 42: 235-242), mice wereinjected subcutaneously (s.c.) with 20 μl of 2.7% solution of formalininto the plantar surface of left hindpaw and placed immediately intoclear PVC observation chambers (23×12×13 cm). Pain behaviour wasquantified by counting the cumulative licking time (s) of the injectedpaw. Measurements were taken during the early phase (0-5 min) and latephase (20-40 min) after formalin injection (Tjolsen A., Berge O. G.,Hunskaar S., Rosland J. H., Hole K. Pain (1992) 51:5-17).

The test compound was administered p.o. or s.c. 5-45 minutes beforeformalin injection in a volume of 10 ml/kg body weight to groups of 10mice per dose. Control group was treated with vehicle.

The orally and subcutaneous administered compounds of the invention canbe found active in this experimental model.

The results expressed as ED₅₀ value, obtained with one compoundadministered at 0.6-20 mg/kg p.o. and s.c., which is representative ofthe entire class of compounds of the invention, and reported in Table 6demonstrates that this compound has a good analgesic activity.

TABLE 6 ED₅₀ (mg/kg) Compound p.o.2-[2-(3-Butoxyphenyl)-ethylamino]-N,N- 14.9 dimethylacetamidehydrochloride (S)-(+)-2-[4-(2-Fluorobenzyloxy)- 29.3benzylamino]-propanamide (ralfinamide)*(S)-(+)-2-[4-(3-Fluorobenzyloxy)- 69.4 benzylamino]-propanamide(safinamide)* *as the salt with methanesulfonic acid

Example 17 Spinal Nerve Ligation Model of Neuropathic Pain

Effects on neuropathic pain are tested in the Spinal Nerve Ligationmodel (SNL) (Kim S. H. and Chung J. M. (Pain (1992) 50: 355-363).

Animals: Adult male Wistar rats weighing 175-200 g were used. Allanimals were housed in groups of 8/10 in a temperature (22±0.5° C.) andrelative-humidity (60-70%) controlled room on a 12-h light/dark cycle(lights on between a 6 a.m. to 6 p.m.) and allowed free access to waterand standard diet for rodents.

Drugs: After measurement of a basal allodynic threshold, test compoundsdissolved in distilled water was administered orally at the doses of0.5-100 mg/kg in a volume of 2 ml/kg. Control rats were treated withvehicle.

Spinal nerve ligation: Neuropathy was produced according to a modifiedmethod described by Kim S. H. and Chung J. M. (Pain (1992) 50: 355-363).Briefly, the animals were anaesthetized with sodium thiopental 35 mg/kgi.p. (plus additional dose if needed) and after the exposure of thedorsal vertebral column from L4 to S2, the exposed L5 and L6 spinalnerves were tightly ligated with 4-0 silk suture, and the incision wasclosed. Rats were allowed to recover after surgery for about 5-14 daysbefore testing.

Mechanical Allodynia: Mechanical allodynia thresholds were determinedaccording to the method of Chaplan et al. (Chaplan S. R., Bach F. W.,Pogrel J. W., Chung J. M. and Yaksh T. L. J. Neurosc. Method. (1994) 53:55-63). Rats were placed in individual plastic boxes of 24×10×15 cm on amesh metal floor and allowed to acclimate for about 30 min beforetesting. The paw withdrawal thresholds of the hind paws of the rats weredetermined in response to probing with 8 calibrated von Frey filaments(Stoelting, Wood Dale, Ill.) with logarithmically incremental stiffnessranging from 0.41 to 15 g (4 to 150 mN). Each filament was appliedperpendicularly to the plantar surface of the ligated paw of rats. Amaximal cut-off of 15 g was used. Withdrawal thresholds was determinedby sequentially increasing and decreasing the stimulus strength(“up-down” method), analyzed by using a Dixon nonparametric test, andexpressed as the mean withdrawal threshold (Dixon W. J. Am. Stat. Assoc.(1965) 60: 967-978). The mechanical allodynia thresholds both in thesham and operated animals was measured before (pre-drug) and at 15, 30,60, 90, 120, 180, 240, 300, 360 and 420 min after p.o. treatment. A 24 hthreshold was also measured in both treatment schedules. The test wascarried out between 9 a.m. and 6 p.m. The observers were blind to theexperimental and treatment conditions.

Thermal hyperalgesia: Thermal hyperalgesia was assessed using theplantar test (Ugo Basile, Varese, Italy). The rats were placed inPlexiglas enclosures on a clear glass plate. With the rat standingrelatively still, a radiant heat source beneath the glass floor wasaimed at the plantar surface of the hind paw, and the withdrawal latencywas measured. Before assessment of thermal hyperalgesia, the intensityof the radiant heat was adjusted to yield a baseline latency of about 20seconds from naïve rats with the cutoff of automatically set at 30seconds to avoid tissue damage.

Orally administered representative compounds of this invention werefound active in this experimental model.

(FIG. 1: Effect of2-[2-(3-butoxyphenyl)-ethylamino]-N,N-dimethylacetamide hydrochloride(NW-3509) orally administered in thermal hyperalgesia in SNL rats.ED50=10.58 mg/kg (6.7-16.6). Values represent the mean+SEM of 10 animalsper group.)

Example 18 Complete Freund's Adjuvant Model of Chronic Inflammatory Pain

Monoarthritis was induced in rats (200 g weight) by an intra-plantarinjection into the left hind paw of 100 μl of complete Freund's adjuvant(CFA, Sigma) containing heat-killed and dried Mycobacterium tubercolosisin a mixture of paraffin oil and an emulsifying agent, mannidemonooleate. A group of naive rats were used as control. The CFAinjection produced an area of localized oedema and inflammation startingfrom few h after injection, with a progressive reduction in themechanical withdrawal threshold.

Each animal was allowed to develop the arthritis over a period of 8-9days before testing. Mechanical Allodynia: Mechanical allodyniathresholds were determined according to the method of Chaplan et al.(Chaplan S. R., Bach F. W., Pogrel J. W., Chung J. M. and Yaksh T. L. J.Neurosc. Method. (1994) 53: 55-63). Rats were placed in individualplastic boxes of 24×10×15 cm on a mesh metal floor and allowed toacclimate for about 30 min before testing. The paw withdrawal thresholdsof the hind paws of the rats were determined in response to probing with8 calibrated von Frey filaments (Stoelting, Wood Dale, Ill.) withlogarithmically incremental stiffness ranging from 0.41 to 15 g (4 to150 mN). Each filament was applied perpendicularly to the plantarsurface of the ligated paw of rats. A maximal cut-off of 15 g was used.Withdrawal thresholds was determined by sequentially increasing anddecreasing the stimulus strength (“up-down” method), analyzed by using aDixon nonparametric test, and expressed as the mean withdrawal threshold(Dixon W. J. Am. Stat. Assoc. (1965) 60: 967-978). The mechanicalallodynia thresholds both in the sham and operated animals was measuredbefore (pre-drug) and at 15, 30, 60, 90, 120, 180, 240, 300, 360 and 420min after p.o. treatment. A 24 h threshold was also measured in bothtreatment schedules. The test was carried out between 9 a.m. and 6 p.m.The observers were blind to the experimental and treatment conditions.

Orally administered representative compounds of this invention werefound active in this experimental model.

Example 19 Acetic Acid-Induced Visceral Pain Model in Mice

Visceral pain is still one of the most common forms of pain, which seeksmedical care. Despite the conventional belief that visceral pain is avariant of somatic pain, it differs in neurological mechanisms andtransmission pathways. Visceral pain is characterized by referralhyperalgesia and also it is not always linked to tissue injury.

The acetic acid-induced visceral pain model is widely used inexperimental research to produce abdominal contractions (Korster R etal, Fed. Pro. (1959) 18: 412; Friese N et al., Life Sci. (1997) 60:625-634) The model consists of intraperitoneal (i.p.) injection of anirritant that induces a syndrome called ‘writhing’, which consists ofcontractions of the abdomen, twisting and turning of the trunk, archingof the back and extension of the hind limbs.

Animals and procedure: Male CD1 mice weighing 25-33 g were used. Eachtreated group was allowed 30 min to habituate to laboratory surroundingsin individual polypropylene transparent boxes. The visceral pain wasscored by counting the number of writhes for 10 min after i.p. injectionof 0.6% acetic acid (10 ml/kg of body weight). The number of writhesafter acetic acid administration were evaluated. Both complete bodystretching (complete writhe) or partial stretching with a clearcontracting of the abdomen (partial writhe) were counted. Separategroups of 10 mice each were administered orally with vehicle (10 ml/kg),or different doses of the tested compound dissolved in vehicle (10ml/kg), 5 min before acetic acid injection. Data are expressed as themean number of writhes during the 10 min observation period.

Orally administered representative compounds of this invention werefound active in this experimental model reducing the number of writhesinduced by acetic acid.

(FIG. 2: Effect of2-[2-(3-butoxyphenyl)ethylamino]-N,N-dimethylacetamide hydrochloride(NW-3509) orally administered at 20 mg/Kg p.o. in acetic acid inducedvisceral pain test. ***p<0.01 vs. vehicle t-test. Values represent themean+SEM of n=10 animals per group. ***p<0.01 Dunnet's test.)

Example 20 Maximal Electroshock Test (MES) in Mice

The maximal electroshock test (MES) is used commonly in the screening ofanti-epileptic drugs in rodent models.

Animals and Apparatus: Male CD1 mice weighing 25 g were used. Theprocedure described by White et al. (White H. S., Woodhead J. H.,Franklin M. R., Swinyard E. A., and Wolf H. H. Antiepileptic Drugs(1995) 4th ed: 99-110, Raven Press, Ltd., New York) was followed. An UgoBasile electroconvulsive generator (Model ECT UNIT 7801) was used todeliver an electrical stimulus sufficient to produce a hindlimb tonicextensor response in at least 97% of control animals. The stimulus wasdelivered intra-aurally through clip electrodes in mice (0.7 s of a 40mA shock, with a pulse train of 80 Hz having a pulse duration of 0.4ms). The acute effect of compounds administered intraperitoneally(i.p.), subcutaneously (s.c.), intravenously (i.v.) or orally (p.o.)5-120 minutes before MES induction were examined and compared with avehicle control group. Ten mice were studied per group. Completesuppression of the hindlimb tonic extensor component of seizures wastaken as evidence of anticonvulsant activity.

The compounds of the invention were administered p.o. or i.v., at thedoses of 0.1-100 mg/kg.

The results, expressed as ED50 values obtained with some compoundsrepresentative of the entire chemical class of the invention, arereported in Table 7 and in Table 8, demonstrate that these compounds areactive as anticonvulsant drugs.

TABLE 7 % protection ED₅₀ Compound 10 mg/kg p.o. (mg/kg p.o.)2-[2-(3-Butoxyphenyl)-ethylamino]-N,N- 100 4.3 dimethylacetamidehydrochloride 2-[2-(3-Butoxyphenyl)-ethylamino]-N- 30 12.1methylacetamide hydrochloride 2-[2-(3-Butoxy-2,6-difluorophenyl)- 1002.0 ethylamino]-N,N-dimethylacetamide hydrochloride2-[2-(3-Pentyloxyphenyl)-ethylamino]- 100 2.8 N,N-dimethylacetamidehydrochloride 2-[2-(3-Butoxy-4-methylphenyl)- 40 n.d.ethylamino]-N,N-dimethylacetamide 2-[2-(3-Butoxyphenyl)-ethylamino]- 606.5 N,N-dimethylpropanamide hydrochloride2-[2-(3-Hexyloxyphenyl)-ethylamino]- 100 4.6 N,N-dimethylacetamidehydrochloride

TABLE 8 MES ED₅₀ Compound mg/kg i.v.2-[2-(3-Butoxyphenyl)-ethylamino]-N,N- 0.2 dimethylacetamidehydrochloride (S)-(+)-2-[4-(2-Fluorobenzyloxy)- 2.7benzylamino]-propanamide (ralfinamide)*(S)-(+)-2-[4-(3-Fluorobenzyloxy)- 4.0 benzylamino]-propanamide(safinamide)* *as the salt with methanesulfonic acid

Example 21 Amphetamine and Chlordiazepoxide-Induced Hyperlocomotion inMice

In this model, mice are treated with a mixture of d-amphetamine plus ananxiolytic dose of the benzodiazepine, chlordiazepoxide (Rushton R,Steinberg H. Combined effects of chlordiazepoxide and d-amphetamine onactivity of rats in an unfamiliar environment. Nature 1966; 211:1312-3;R. Arban, G. Maraia, K. Brackenborough, L. Winyard, A. Wilson, P.Gerrard, C. Large, Evaluation of the effects of lamotrigine, valproateand carbamazepine in a rodent model of mania Behavioural Brain Research,158: 123-132). The model has been claimed to mimic some aspects of maniain bipolar disorder Importantly, the hyperactivity induced by themixture of d-amphetamine and chlordiazepoxide could be prevented byprior administration of the established mood stabilizer, lithium, aswell as other mood stabilizers drugs (e.g. magnesium valproate andcarbamazepine). Therefore, this model has face and predictive validityas a model of bipolar disorder and represents a valuable tool todetermine, if a test compound could be a potential mood stabilizer drugcandidate.

Amphetamine (AMP) (2.5 mg/kg) plus chlordiazepoxide hydrochloride (CDZ)(3 mg/kg/ip) were administered to male Albino Swiss mice (25-32 g) in avolume of 10 ml/kg. The locomotor activity was recorded using Opto-M3System (Columbus Instruments) which is multi-channel activity monitor.Opto-M3 system has 10 infrared emitters and respective amount ofreceivers (0.5″ beam spacing), attached to the PC computer andcalculating both ambulatory activity and total counts. Thus the systemdifferentiates forward locomotion (ambulation) from stereotyped likemovement (total counts). Mice were pretreated with the test compound(0.5-20 mg/kg) and 10 min later, with AMP (2.5 mg/kg) or AMP jointlywith CDZ (3 mg/kg). After successive 30 min the mice were treated againwith the same dose of the test compound and were placed individually inthe motor activity cages. The locomotor activity (ambulation and totalactivity count) was evaluated for 30 min Each group consisted of 8-10mice.

Statistical analysis: the data were evaluated by an analysis of variance(ANOVA), followed, when appropriate, by individual comparison with thecontrol using Dunnett's test.

Results show that amphetamine and amphetamine-chlordiazepoxide (CDZ)administration induced a significant increase in locomotor activity.

Orally administered representative compounds of this invention werefound active in this experimental model decreasing amphetamine andamphetamine-chlordiazepoxide induced hyperactivity.

(FIG. 3: Ambulation over 30 min.

a) chlordiazepoxide (3.0 mg/Kg ip); b)2-[2-(3-butoxyphenyl)ethylamino]-N,N-dimethylacetamide hydrochloride(NW-3509) (20.0 mg/Kg po); c) amphetamine (2.5 mg/kg i.p.).

Amphetamine alone and the mixture amphetamine+chlordiazepoxidesignificantly increased locomotor activity **p<0.001 Dunnet's test vs.vehicle.

2-[2-(3-Butoxyphenyl)ethylamino]-N,N-dimethylacetamide hydrochloride(NW-3509) reduced hyperactivity induced by both amphetamine alone and bythe mixture of amphetamine and chlordiazepoxide in a statisticalsignificant way ##p<0.001 Dunnet's test vs. amphetamine oramphetamine+chlordiazepoxide. Data represent the mean+SEM of n=10 miceper group.)

Example 22 Morris Water Maze Test (Amnesia Induced by Scopolamine inRat)

The method which detects antiamnesic activity, follows that described byMorris (Learn. Motiv., 12, 239-260, 1981). The Morris Maze consists of acircular water tank (Diameter=150 cm) filled with water and maintainedat 26-28° C. with an escape platform (Diameter=15 cm) 18 cm from theperimeter and always in the same position 1.5 cm beneath the surface ofthe water. The water is made opaque by addition of a non-toxic coloringagent (e.g. milk powder) rendering the platform invisible.

The animals are given 4 training sessions over 4 consecutive days (Day 1to Day 4). Each training session consists of 4 trials in the MorrisMaze, each separated by 1 minute. For each trial the animal is placed inthe maze at one of four starting points equally distributed around themaze and allowed to find the escape platform. The animal is left on theescape platform for 60 seconds before being returned to its home cage.If the animal does not find the platform within 120 seconds theexperimenter removes it from the water and places it on the platform for60 seconds before replacing it in its home cage. During the 4 trials theanimals start the maze once from each starting point in a randomlydetermined order per animal.

The trials are video-recorded and the behaviour of animals is analyzedusing a video-tracking system (Panlab: SMART). The principal measuretaken is the escape latency at each trial. Additional measures are theswim speed and distance travelled.

Scopolamine-treated animals (0.5 mg/kg i.p., administered 30 minutesbefore each session) show amnesia in this task as indicated by thefailure to reduce their escape latencies from trial to trial and fromsession to session. A probe test will be performed on Day 5. Theplatform is removed from the maze and the animal allowed to swim freelyin the maze for 60 seconds. The principal measure taken is the timespent in the target quadrant (i.e. that previously containing theplatform), which is compared with the time spent in the other quadrantsand with the value expected by chance (i.e. 25% of probe test duration).Twelve scopolamine-treated rats are studied per group. The experimentalso includes a normal control group receiving saline instead ofscopolamine. The test substance is evaluated at 3 doses, administeredp.o. 5-60 minutes before the each training session and the probe trial,i.e. 30 minutes before scopolamine, and compared with a vehicle controlgroup. The experiment will therefore include 5 groups.

Data are analyzed by comparing treated groups with scopolamine controlusing unpaired Student's t tests.

Orally administered representative compounds of this invention werefound active in this experimental model.

Example 23 Cognition Model-Novel Object Recognition Test

The object recognition test consists of a sample trial and a choicetrial separated by an inter-trial interval (ITI). On the sample trial,two identical objects are presented. On the choice trial, one of theobjects presented in sample trial (termed as familiar objects) isreplaced by a new object. Rats are able to discriminate between thefamiliar object and the new object when the ITI is 1 h or less, but notwith a 24 h ITI (Deschaux O, et al, 1997, Neurosci. Lett. 222, 159-162;Ennaceur A, et al, 1989., Behav. Brain Res. 33, 197-207; Puma C, Bizot JC, 1998, Neurosci. Lett. 248, 183-186.). Therefore, the objectrecognition task with a 1 h ITI allows to detect the amnesic effect of adrug and whether this amnesic effect is reduced by another drug. Theobject recognition test with a 24 h ITI allows to detect a drug-inducedenhancement of memory. Numerous studies have been conducted in order toassess memory effects of drugs. For example, past studies demonstratedthat nicotine improves memory in the 24-h ITI condition and reducesscopolamine-induced amnesia in the 1-h ITI condition.

Methods: The object recognition task is performed as described in BizotJ C, et al, 2005 Prog Neuropsychopharmacol Biol Psychiatry. 29:928-935

Subjects: Naive 8-week old male Wistar rats were used

Apparatus: The apparatus of the object recognition task is an open boxmade of grey opaque Plexiglas (40 cm L, 40 cm W, 40 cm H). The objectsto be discriminated (3.5 cm L, 6 cm H) differ in both color and shape.They are a white door button round shape and a grey door button starshape. Apparently, they have no natural significance for rats and theyhad never been associated with reinforcement. In order to rule out thepossibility of scent traces left on the objects and therefore thedependency of the recognition capacity of rats on the olfactory cue, theobjects and the ground of the box are washed with clear water and driedbetween each trial. A video camera is fixed to the ceiling above the boxto monitor the animals' activity.

Experiments take place over 2 days (scopolamine-induced amnesia) or 3days (natural forgetting). The test consists of a 15-min habituationtrial (day 1), a 3-min sample trial (day 2) and a 3-min choice trial(day 2 or day 3).

Scopolamine-induced amnesia experiment. This experiment is conducted onrats randomly subdivided in different groups (n=12/group) which receive:

-   -   control group: 1 intraperitoneal (IP) injection of saline 30 min        before the test (sample trial) 1 per-os (PO) injection of saline        15 min before the test and.    -   Scopolamine group: 1 IP injection of scopolamine (0.1 mg/kg) 30        min before the test and 1 PO injection of saline 15 min before        the test (sample trial)    -   Scopolamine+Nicotine group: 1 IP injection of scopolamine (0.1        mg/kg) 30 min before the test and 1 IP injection of nicotine        (0.4 mg/kg) 20 min before the test (sample trial)    -   Scopolamine+representative tested compound groups: 1 IP        injection of scopolamine (0.1 mg/kg) 30 min before the test        (sample trial) and 1 PO injection of different doses of the        tested compound 15 min before the test (sample trial).

On Day 1 The rat is allowed to explore the apparatus for 15 min(Habituation trial). On day 2 the rat is placed in the apparatus withtwo identical objects presented in two corners of the box for 3 min(sample trial). After 1 hr the rat is placed in the apparatus with twoobjects; one of the objects presented in sample trial (termed asfamiliar objects) is replaced by a new object. (Choice trial).

Natural forgetting experiment: This experiment is conducted on ratsrandomly subdivided in different groups (n=12/group) which receive

-   -   Control group: 1 intraperitoneal (IP) injection of saline 20 min        before the test and 1 per-os (PO) injection of saline 15 min        before the test.    -   Nicotine group: 1 IP injection of nicotine (0.2 mg/kg) 20 min        before the test and 1 PO injection of saline 15 min before the        test.    -   Representative tested compound group: 1 IP injection of saline        20 min before the test and 1 PO injection of tested compound at        various doses 15 min before the test.

On Day 1 The rat is allowed to explore the apparatus for 15 min(Habituation trial). On day 2 the rat is placed in the apparatus withtwo identical objects presented in two corners of the box for 3 min(sample trial). Twenty four hours later (day 3) the rat is placed in theapparatus with two objects; one of the objects presented in sample trial(termed as familiar objects) is replaced by a new object.

Data: The basic measurement is the time spent by the rats in exploringthe objects during the sample trial and during the choice trial. Objectrecognition task indices include the following parameters:

-   -   The total exploration time in the sample trial,    -   The total exploration time in the choice trial.    -   The difference of exploration time between the new object and        the familiar object in the choice trial (N−F).    -   The discrimination index, that is 100×(N−F) divided by the total        exploration time in the choice trial.

Data are analysed by ANOVA and Student's t-test.

Orally administered representative compounds of this invention werefound active in this experimental model reducing scopolamine-inducedamnesia in the object recognition task with a 1-h inter trial interval(1 hr ITI) and improving memory in a natural forgetting situation, i.e.in the object recognition task with a 24 hours inter trial interval (24hr ITI).

Example 24 Depression Test: Tail Suspension Test in the Mouse

Tail-suspension test is one of the so-called “behavioural despair”models and are used for the screening of antidepressant drugs. They arebased on a common phenomenon: a normal animal submitted to a non-solubleaversive situation alternates between agitation and immobility. Thereason of agitation is searching, it is highly energy consuming, whilethe purpose of immobility is energy conservation Animals afterantidepressant treatment struggle more even in desperate situation, andthey spend less time with immobility. Some aspects of neuroticdepression can be studied with the aid of these models.

The present method detects antidepressant and anxiolytic activity andfollows that described by Stem et al (Psychopharmacology, 85, 367-370,1985). Rodents, suspended by the tail, rapidly become immobile.Antidepressants decrease the duration of immobility, whereastranquillizing agents increase the duration of immobility. The behaviourof the animal is recorded during 6 minutes automatically using acomputerized device (Itematic-TST) developed by Stem et al (Prog.Neuropsychopharmacol. Exp. Psychiatry, 11, 659-671, 1987). 6 mice arestudied simultaneously. Two parameters are recorded:

-   -   Duration of immobility: this parameter is analogous to that used        in the “behavioural despair” test (Arch. Int. Pharmacodyn.        Ther., 229, 327-336, 1977).    -   Power of movements: this parameter, based on the energy expended        by the animal, is independent of the duration of activity.

10 mice are studied per group. The test is not performed blind but therandomization schedule generated by the Itematic-TST ensures ahomogeneous distribution of the treatments both in time and in theposition of each animal in the apparatus. The test substance isevaluated at 3 doses, administered p.o. 5-60 minutes before the test,and compared with a vehicle control group. Imipramine (128 mg/kg p.o.)and diazepam (8 mg/kg p.o.), administered under the same experimentalconditions, are used as reference substances.

The experiment therefore includes 6 groups. Data are analyzed bycomparing treated groups with vehicle control using unpaired Student's ttests.

Orally administered representative compounds of this invention werefound active in this experimental model.

Example 25 Cognitive Impairment in Schizophrenia Method

Cognitive impairment is often associated with schizophrenia and it hascome to be recognized as a core element of the disorder, bearing onpatient's recovery and re-integration into society.

Particular interest has recently attracted a pharmacological model ofcognitive dysfunctions in schizophrenia, which is based on the effectsof glutamate NMDA receptor antagonists such as phencyclidine (PCP) andketamine (Javitt et al., Am. J. Psychiatry, 1991 October, 148(10),1301-1308) which impair attention and increase “impulsivity” and“compulsive” perseveration in mice performing a complex task (Greco etal., Psychofarmacology (Berl) 2005 April 179(1), 68-76).

Materials and Methods

Animals:

Male DBA/2N mice (Charles River, Italy) were used. The mice weighed25-30 g at the start of the experiments, and were housed undertemperature-controlled conditions (21° C.) with a 12 h light 12 h darkcycle (light on 7:00 am-7:00 pm). Food (Rieper, Italy) was available adlibitum. The animals had two hours of access to water at the end of eachday's testing.

The Five-Choice Serial Reaction Time Task Apparatus:

The test apparatus consisted of four 21.6×17.8×12.7 cm chambers (MedAssociates Inc. USA), as previously described (Greco et al.,Psychofarmacology (Berl), 2005 April 179(1), 68-76). Stimuli andrecording of responses, were managed by a SmartCtrl™ Package 8 In/16 Out(Med Associates Inc. USA) with additional interfacing by MED-PC forWindows (Med Associates Inc. USA). The running program for the 5-CSRTtask was custom-written.

Behavioural Procedures: Habituation to Liquid Reinforcer and Nose-Pokingin the Holes.

Mice were handled for one week and their body weight recorded. They werethen water-deprived by allowing them 2-h access to water in the earlyevening until their body weight had stabilised (8 days). Then, over thenext two days the mice were habituated in their home cages to thereinforcer (10% sucrose solution) used afterwards in the operantprocedures. On the following two days mice were habituated to theoperant boxes. During this stage, 10% sucrose solution was available ina small bowl placed below the receptacle hole of the box. First, micehad to learn that every 5 sec the liquid reward was available in a smallcup in the receptacle hole. During this period head entries wererecorded. During the next period, mice were trained to poke their nosesinto the illuminated holes Immediately after a poke in the waterreceptacle a LED at the rear of one of the holes was turned on. Anose-poke in the lighted hole extinguished the light stimulus and theliquid dipper provided a 0.01 mL liquid reward in the receptacle hole.Any response in one of the other four holes had no consequence and wasnot recorded. The light stimulus was presented in all five holes inrandom order. A mouse was switched to the 5-CSRT task after it hadcompleted at least 50 rewarded nose-poke trials in one 30-min session.

The Five-Choice Serial Reaction Time Task.

The start of the session was signalled by illumination of thehouse-light and the delivery of a 0.01 mL liquid reward. Nose poking inthe receptacle hole began the first trial. After a fixed delay (theinter-trial interval, ITI), the LED at the rear of one of the holes cameon for a short period. The LED stimulus was presented the same number oftimes in each hole during a complete session, with the order ofpresentation randomised by the computer. While the light was on, and fora short period afterwards (the limited hold), responses in the hole thatwas illuminated (correct response) resulted in the liquid reward.Responses in the holes that had not been illuminated (incorrectresponses) or failure to respond within the limited hold (omissions)caused the house-lights to be turned off for a short period (time out).Responses in the holes while the house-light was off restarted the timeout. After the delivery of the liquid reward, or at the end of time out,the mouse started the next trial by poking its nose into the receptaclehole. Responses made in the holes after a correct response(perseverative responses), or after the end of time out beforenose-poking into the receptacle hole, resulted in a period of time out.Responses in the holes during the ITI (anticipatory responses) alsoresulted in a period of time out. After anticipatory responses anose-poke into the receptacle hole restarted the current trial. Eachdaily session consisted of 100 trials or 30 min of testing, whicheverwas completed sooner, after which all lights were turned off and furtherresponses had no effect. In the first session of the test schedule, thestimulus and limited hold each lasted 1 min and, depending on individualperformance, they were progressively reduced to 1 sec. The stimulusduration was reduced in the following sequence: 60, 30, 10, 5, 2.5, 2,1.5 and 1 sec (baseline). The ITI and time out both lasted 2 sec duringthe first session and the ITI was raised to 5 sec in subsequentsessions; time out was not changed. Throughout the whole period oftraining and experiments each mouse had one session per day on a 5-CSRTtask.

Drugs and Treatment Schedules.

The test compound is dissolved in water and is administeredintraperitoneally (i.p.) at the dose of 10 mg/kg. Five minutes after thetreatment mice were injected with vehicle (saline) or PCP (1.5 mg/kg)and 10 min later they started the test session. In each experiment thevarious combination of the test compound with vehicle or PCP areadministered according to a Latin-square design. At least 48 h are leftbetween the drug testing days. During these intervening days the miceare tested on the 5-CSRT task to re-establish baseline performance andto check for any residual effects of drugs.

Statistical Analysis:

the main dependent variables selected for analysis are: (a) thepercentage of correct responses (total correct responses/totalcorrect+total incorrect responses×100); (b) percentage of omissions(total omissions/total correct responses+total incorrect responses+totalomissions×100); (c) the number of anticipatory responses in the holesduring the ITI; (d) the number of perseverative responses in the holesafter a correct response. Correct responses and omissions, aspercentages, are transformed according to the formula 2 arcsin(SQRT (%X/100)), to normalize the distributions in accordance with the ANOVAmodel (Winer, 1971).

The effects of the test compound (n=12) on PCP induced deficits in the5-CSRT task were analysed independently by a within subjects 2×2 ANOVAwith factors Drug (test compound) and PCP. Subsequently the treatmentgroup means are compared using a post-hoc Tukey-Kramer test. Statisticalsoftware (SAS Institute Inc., USA) was run on Micro VAX 3500 computer(Digital, USA).

PCP causes a profound effect on attentional performance of DBA/2N miceincreasing anticipatory and perseverative responses.

Representative compounds of this invention, administered 10 mg/Kg i.p.,reversed PCP-induced increase in anticipatory and perseverativeresponses, supporting the use of this kind of compounds for thetreatment of psychiatric disorders.

Example 26 Prepulse Inhibition of Startle in Mice and Rats

Prepulse inhibition (PPI) is a cross-species phenomenon (ie, it ispresent in mammals ranging from mice to humans), yet it is relativelyabsent among schizophrenic patients. The reduced ability to filter outamong irrelevant auditory stimulation is a characteristic thought tocontribute to certain manifestations of these conditions includinginattention, distractibility, and cognitive deficits. The PPI procedureis used to assess the subject's ability to “gate” or filterenvironmental information. In the acoustic (startle model) ofsensorimotor gating a weak acoustic stimulus (prepulse) decrease thereflexive flinching response (startle) produced by a second, moreintense, stimulus (the pulse). Drugs like dizocilpine (MK-801) oramphetamine disrupt PPI and represent an animal model of schizophrenia.Antipsychotic drugs are able to prevent PPI deficit. The test is quiteuseful to screen potential antipsychotic drugs.

Similarly some strains of mice such as the DBA/J display a spontaneousimpairment of PPI that can be reversed by antipsychotic drugs and arealso used as animal model of schizophrenia.

PPI in rat: Wistar or Sprague-Dawley rats (weighing 200 to 300 g) or3-week-old DBA/2J mice were used. The startle apparatus (San DiegoInstruments, CA) consisted of 12 plastic transparent cages, placedindividually in a sound-proof cabinets, equipped with a movable platformfloor attached to a sensor recording vertical movements of the platform.Startle reaction was evoked by acoustic stimuli delivered by aloudspeaker suspended above the cages and connected to an acousticgenerator. The transient force resulting from the movements of theplatform evoked by the startle reaction was recorded with a PC computerduring a recording window of 200 ms measured from the onset of theacoustic stimulus, digitalized and stored in the computer for furtherevaluation. The amplitude of the startle response was measured duringthe whole recording window (200 ms) and an average value of amplitudewas taken for further evaluation. The control and treated rats wereplaced in the testing cages individually. After 5 min of habituation(background white noise, 65 dB), two types of acoustic stimuli were usedin random order: acoustic stimulus alone [120 dB, 40 ms, (P)] or thestimulus proceeded by a prepulse [75 dB, 20 ms (PP)] applied 100 msbefore the stimulus. During each experimental session 20 trials of eachtype were presented with interstimulus interval of 20 s. The amplitudeswere averaged for each individual animal, separately for both types oftrials (stimulus alone or stimulus preceded by the prepulse). Thepercent PPI was calculated with the following formula: 100−[(meanstartle amplitude for prepulse+pulse trials/mean startle amplitude forpulse alone trials)×100] i.e. 100−(PP/P)×100. A high value of thecalculated % PPI indicated that the prepulse inhibited the response to apulse stimulus, whereas a low value indicated weaker inhibition byprepulse. Deficits of sensorimotor gating was induced by MK-801 (0.2mg/kg) given ip 5 min before test or amphetamine (2.5 mg/kg) given sc 10min before test. Orally administered representative compounds of thisinvention were found active in this experimental model, reversing PPIdeficit induced by MK-801 (FIG. 4) or by amphetamine.

PPI in DBA mice: Male 3-week-old DBA/2J and C57BL/6J mice were used.

Detection of acoustic startle response and PPI was performed asdescribed in Bortolato et al Psychopharmacology, 2007, October; 194(3):361-9.

In each experiment, mice were assigned to receive either compounds ofthe invention or vehicle and were tested in the PPI session using abetween-subjects design.

The percent PPI was calculated with the following formula: 100−[(meanstartle amplitude for prepulse+pulse trials/mean startle amplitude forpulse alone trials)×100] i.e. 100−(PP/P)×100. The magnitude of theacoustic startle response was calculated as the average response to allof the pulse-alone trials, excluding the first and last blocks of fivepulse-alone trials presented.

Orally administered representative compounds of this invention werefound active in this experimental model reducing PPI deficit of BDA/2Jmice.

(FIG. 4: Effect of2-[2-(3-butoxyphenyl)ethylamino]-N,N-dimethylacetamide hydrochloride(NW-3509) in MK-801 PPI disruption in rats. Data are expressed asmean+SEM of n=10 animals. p<0.01 indicate statistically significantdifference between vehicle and MK-801. *p<0.05 **p<0.01 indicatestatistically significant differences between vehicle+MK-801 treatedanimals and animals treated with2-[2-(3-butoxyphenyl)ethylamino]-N,N-dimethylacetamide hydrochloride(NW-3509).

Analysis of variance followed by Dunnet's test.)

Example 27 Paradoxical Sleep Deprivation in Rats

The psychopathological and neurobiological relationships between sleepand psychotic phenomena have been evidenced by a number of classicalclinical observations and psychological reports. Schizophrenic patientsexhibit severe insomnia, together with a number of structuralalterations of sleep architecture, such as a reduction in REM sleeplatency and duration, as well as a decrease in SWS (Slow Wave Sleep)time. Numerous studies have demonstrated that prolonged sleepdeprivation is indeed conducive to a number of transient psychologicaldisorders, including impaired verbal constructions, disorganizedthought, depersonalization and perceptual changes, ranging from minordisturbances and abnormal bodily sensations to complex acoustic andvisual hallucinations. Typically, these disorders are indistinguishablefrom psychotic phenomena, but are normally extinguished following aprolonged recuperative sleep. Several studies have documented thatfollowing a period of forced sleep deprivation, rats exhibit for alimited time a paradoxical array of behavioral changes, such asstereotyped behavior and hyperactivity. Moreover, sleep deprivation inrats enhances startle reaction and disrupts PPI, this effect istime-dependent and reversible by antipsychotic drugs (Gessa, G L et al.(1995) European Neuropsychopharmacology 5, 89-93). Such phenomena arecommonly interpreted as relevant to psychosis, as they are alsotypically produced by psychotomimetic agents in rats. The model of sleepdeprivation in rats can be considered a model of mania.

Animals: Sprague-Dawley rats (weighing 200 to 300 g) were used.

Methods: The procedure to induce paradoxical sleep deprivation (SD) inrats is based on the platform method (modified from Jouvet, et al(1964). Journal of Physiology (Paris), 56, 381). Rats were kept on asmall Plexiglas platform (7 cm in diameter) within a deep tank filledwith water. Each platform was surrounded by water up to 1 cm, beneaththe surface. Chow pellets and water bottles were located on a grid onthe top of the tank. During the whole SD study, the temperature of theexperimental room and the water inside the tank were maintained at 23±1°C. Control rats are placed in the experimental room, either in theirhome cages or on water tanks equivalent to those used for SD, but with a12 cm-diameter platform, which allows them to reach REM sleep withoutfalling into the water. At the end of the SD period (72 hr), rats wereimmediately dried out and placed either in the startle chambers (for PPImeasurement) or in the activity cages (hyperactivity) for behavioraltesting. The water in the tank was changed daily throughout the SDperiod. Control rats were maintained in the same room as thesleep-deprived rats for the duration of their SD. Compounds to be testedwere given before PPI or behavioural testing.

Data Analysis: For each animal, the mean startle amplitudes for thefirst and the second halves of the second period of the session (blocks,six pulse-alone trials each) were analyzed with a two-way or three-wayanalysis of variance (ANOVA), with pretreatment (where present)treatment as between-subjects factors and blocks as repeated measures.The percent PPI was calculated with the following formula: 100−[(meanstartle amplitude for prepulse+pulse trials/mean startle amplitude forpulse alone trials)×100] i.e. 100−(PP/P)×100 and analyzed in multifactorANOVAs (with specific design and comparisons noted below for eachexperiment) with the different combinations of injections forpretreatment and treatment as between-subjects factors and trial typesas repeated measures. Post hoc analyses were performed using Tukey'stest. Locomotor activity data were analysed using the one- or two-wayanalysis of variance (ANOVA) for repeated measures when appropriate,followed by Tukey's test as post-hoc tests.

Orally or intraperitoneally or subcutaneously administeredrepresentative compounds of this invention were found active in thisexperimental model reducing PPI deficit and hyperactivity induced bysleep deprivation.

Example 28 Cocaine-Induced Behavioural Sensitization Test

Drug addiction is a pathological behaviour characterized by compulsivedrug seeking and intake. One animal model of these behavioural changesis the long-lasting increase in locomotor activity induced by repeatedadministration of psychostimulant drugs in rodents (Robinson T. E. andBerridge K. C. Brain Res. Brain Res. Rev. (1993) 18, 247-91) known asdrug-induced behavioural sensitization. The effect of test compoundswere evaluated in a model of cocaine-induced behavioural sensitizationin rat.

Locomotor activity apparatus: Male Wistar rats weighing 200-250 g uponarrival were used. Locomotor activity was measured in sixteen identicalmetal wire hanging cages each measuring 36 cm (L)×25 cm (W)×20 cm (H).Each cage contained two sets of infrared emitter-detector photocellspositioned along the long axis 1 cm above the grid floor and 8 cm fromthe front and back of the cage. Background noise was provided by a whitenoise generator. Movement within the cages produced photocellinterruptions, which were automatically recorded by an IBM-compatiblecomputer.

Sensitization procedure and treatment: Animals were habituated to thelocomotor activity chambers for 2-3 consecutive days before theexperiment. Rats received 5 daily i.p. injections of cocaine (15 mg/kg)or saline and either the test compound (0.1-100 mg/kg) or its vehicleand locomotor activity was recorded for 3 h. Ten days after the lastinjection of cocaine or saline (day 15), the animals were challengedwith 15 mg/kg of cocaine in absence of the test compound and locomotoractivity was again monitored for 3 h.

By the fifth day of treatment with cocaine, animals pretreated i.p. withvehicle showed an increased locomotor response (20% higher then thefirst day, p<0.05). Ten days after the last injection of cocaine orsaline, the animals were challenged with 15 mg/kg of cocaine in absenceof the test compound and locomotor activity was again monitored for 3 h.The rats previously treated with cocaine and that had not received thetest compound are expected to show an increased locomotor activityresponse to cocaine (30% higher then first day, p<0.05). If the ratsthat had been pretreated with the test compound during the 5 day-cocainetreatment did not show an increase in locomotor activity the testcompound is considered to have an effect in preventing psychostimulantdrugs addiction. (Koob G. F., Sanna P. P., Bloom F. E. Neuron (1998) 21:467-476; Robinson T. E., Berridge K. C. Brain Res Brain Res Rev (1993)18: 247-291)

Statistical analysis: Data (total number of beam breaks in 3 hours) wereanalyzed using a two way ANOVA with repeated measures on one factorincluding the four experimental groups (i.e., saline/vehicle,saline/test compound, cocaine/vehicle and cocaine/test compound) and twotime points (day 1 and day 5) followed by a simple effects analysis. Asecond two way ANOVA with repeated measures on one factor was used tocompare day 1 and the challenge day followed by a Newman-Keuls post hoctest.

Orally administered representative compounds of this invention werefound active in this experimental model.

Example 29 Acute Bladder Irritation by Acetic Acid in Rats

Experiments were performed using adult anesthetized female SpragueDawley rats (170-200 g). A catheter (PE-50) was inserted via a midlineabdominal incision into the bladder through the bladder dome, and thenintravescical pressure was measured to monitor bladder activity duringcontinuous infusion of 0.15% acetic acid. Continuous intravescicalinfusion of acetic acid irritates the bladder and reduces theintercontraction intervals (ICI) in anesthetized rats. ICIs, maximalcontraction pressure, and pressure thresholds inducing reflex bladdercontraction were measured before and after intravescical infusion ofacetic acid in rats treated with compounds of the invention.

Intraperitoneally or intravenously administered representative compoundsof this invention were found active in this experimental model.

Example 30 Intermediate Bladder Irritation by Cyclophosphamide (CYP) inRats

Experiments were performed using both adult awake and anesthetizedfemale Sprague Dawley rats (170-200 g). Chemical cystitis was induced byCYP, which is metabolized to acrolein, an irritant eliminated in theurine. CYP (150 mg/kg/i.p.) was administered one day before theexperiment. Pre-treatment with CYP causes bladder irritation and veryfrequent voidings with an ICI of about 150-200 seconds between voids.

Intraperitoneally or intravenously administered representative compoundsof this invention increased the ICI in both awake and anesthetized ratsused in this experimental model.

Example 31 Migraine Test in Rats

Animals and surgery: Male Wistar rats (250-350 g) were anesthetized withsodium pentobarbital (50 mg/kg i.p.) dissolved in saline.

The trachea and left femoral artery were cannulated for artificialventilation (55 strokes/min) and for measurement of mean blood pressure(MBP) respectively. The femoral vein was cannulated for the intravenousadministration of test agents.

Body temperature was maintained at 37-38° C. by automatic control of aheating pad. Animals were placed in a stereotaxic frame and alongitudinal incision was made in the scalp. A burr hole was drilled inthe skull and a stainless steel bipolar electrode (Plastic One MS 306)was lowered into left ophthalmic branch of the trigeminal ganglion (3.8mm dorsal to bregma, 2.5 mm lateral from the midline and 9.5 mm belowthe dural surface) and secured with dental cement. Correct placement ofthe electrode was confirmed by a brief electrical stimulation, whichcause movement of the jaw due to activation of the trigeminal fiber.Following removal of the brain, the correct position of the electrodeinto the fiber, was visually checked at the end of each experiment.

A second hole was drilled ipsilateral of the electrode (1.5 mm rostralto bregma, and 1.5 mm lateral from the sagittal suture) and a needleprobe (tip diameter 0.8 mm) of a laser doppler flowmeter was fixedpointing with its tip onto a branch of the middle cerebral artery (MCA)and Cerebral Blood Flow (CBF) change recorded on-line by the PeriFlux4001 Laser Doppler system.

Artefacts of the laser Doppler reading during electrical stimulation ofthe trigeminal ganglion due to muscular movements were prevented by abolus of i.v. injection of the neuromuscular blocker pancuronium bromide(0.6 mg/kg i.v.).

Anaesthesia and neuromuscular blockade were maintained all over theexperiment with an infusion of sodium pentobarbital and pancuronium(12.5 mg/kg/h+2.4 mg/kg/h, respectively).

Experimental protocol: At the end of the surgery, a pause of thirtyminutes was taken in order to stabilize the measured parameters.

Rest CBF was increased by electrical stimulation with rectangular pulseof 0.5 ms length, 1-10 Hz, 0.5-1 mA for periods of 30 s. After twoaveraged pre-drug stimulations, vehicle or drugs were administered.

Intravenously administered representative compounds of this inventionreduced the increase in blood flow induced by trigeminal stimulation.

The invention claimed is:
 1. A method for modulating voltage gatedsodium and/or calcium channels, comprising the step of administering toa patient in need thereof an effective amount of a compound of formula(I):

wherein: X is —O—; Y is hydrogen, —OH or —O(C₁-C₄)alkyl; Z is ═O or ═S;R is —(C₃-C₁₀)alkyl; ω-trifluoro(C₃-C₁₀)alkyl; R₁ and R₂ are,independently, hydrogen, hydroxy, (C₁-C₈)alkoxy, (C₁-C₈) alkylthio,halo, trifluoromethyl or 2,2,2-trifluoroethyl; or one of R₁ and R₂ isortho to R—X— and taken together with the same R—X—, represents a

group where R₀ is —(C₂-C₉)alkyl; R₃ and R′₃ are, independently, hydrogenor —(C₁-C₄)alkyl; R₄ and R₅ are, independently, hydrogen or—(C₁-C₄)alkyl; or R₄ is hydrogen and R₅ is —CH₂—OH, —CH₂—O—(C₁-C₆)alkyl,—CH(CH₃)—OH, —(CH₂)₂—S—CH₃, benzyl or 4-hydroxybenzyl; or R₄ and R₅,taken together with the adjacent carbon atom, form a (C₃-C₆)cycloalkylresidue; R₆ and R₇ are, independently, hydrogen or —(C₁-C₆)alkyl; ortaken together with the adjacent nitrogen atom form a 5- or 6-memberedmonocyclic saturated heterocycle, optionally containing —O—, —S— or—NR₈—, wherein R₈ is hydrogen or (C₁-C₆)alkyl; its pharmaceuticallyacceptable salts and the isolated optical isomers or mixtures of opticalisomers of the compound and their pharmaceutically acceptable salts,wherein a disease treatable by such modulation is a gastrointestinaldisorder.
 2. The method of claim 1, wherein said gastrointestinaldisorder is caused by a dysfunction of a voltage-gated sodium channel.3. The method of claim 1, wherein said gastrointestinal disorder iscaused by a dysfunction of a voltage-gated calcium channel.
 4. Themethod of claim 1, wherein said patient is sensitive to unwanted sideeffects of MAO inhibitory effects.
 5. The method of claim 1, wherein thecompound is administered with at least one other therapeutic agent. 6.The method of claim 1, wherein the effective amount of the compoundadministered to the patient in need thereof (i) does not exhibit anyMAO-inhibitory activity or (ii) exhibits a reduced MAO-inhibitoryactivity.
 7. The method of claim 1, wherein the compound is2-[2-(3-butoxyphenyl)-ethylamino]-N,N-dimethylacetamide or apharmaceutically acceptable salt thereof.
 8. The method of claim 1wherein the gastrointestinal disorder is selected from the groupconsisting of ulcerative colitis, Crohn's disease, ileitis, proctitis,celiac disease, enteropathies, microscopic or collagenous colitis,eosinophilic gastroenteritis, or pouchitis resulting afterproctocolectomy and post ileonatal anastomosis, pylorospasm, nervousindigestion, spastic colon, spastic colitis, spastic bowel, intestinalneurosis, functional colitis, mucous colitis, laxative colitis,functional dyspepsia, atrophic gastritis, gastritis varialoforme,ulcerative colitis, peptic ulceration, pyresis, damage to the GI tractby Helicobacter pylori, gastroesophageal reflux disease, gastroparesis,such as diabetic gastroparesis, non-ulcerative dyspepsia (NUD); emesis,diarrhoea, and visceral inflammation.